Compositions and methods for silencing SMAD4

ABSTRACT

The present invention provides compositions comprising therapeutic nucleic acids such as interfering RNA (e.g., dsRNA such as siRNA) that target SMAD4 gene expression, lipid particles comprising one or more (e.g., a cocktail) of the therapeutic nucleic acids, methods of making the lipid particles, and methods of delivering and/or administering the lipid particles (e.g., for treating anemia of inflammation in humans).

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application61/579,601 filed Dec. 22, 2011, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is TEKM_(—)075_(—)01US_ST25.txt. The text file is 6KB, was created on Dec. 21, 2012, and is being submitted electronicallyvia EFS-Web.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates to compositions comprising therapeuticnucleic acids that target SMAD4 gene expression, lipid particlescomprising one or more of the therapeutic nucleic acids, methods ofmaking the lipid particles, and methods of delivering and/oradministering the lipid particles (e.g., for treating anemia ofinflammation in humans).

II. Description of the Related Art

Anemia of inflammation (sometimes called anemia of chronic disease, oranemia of inflammatory response) is a form of anemia seen in chronicillness (e.g. chronic infection, chronic immune activation, ormalignancy), and is the most common anemia found in hospitalizedpatients. Anemia of inflammation is abbreviated herein as “AI”. Althoughthere may be more than one underlying cause of AI, it appears likelythat the syndrome is largely the result of the production of hepcidin, aprotein that regulates human iron metabolism. It is believed that inresponse to inflammatory cytokines, such as IL-6, the liver producesincreased amounts of hepcidin which, in turn, prevents the proteinferroportin from stimulating release of stored iron.

Ideally, AI is resolved by successful treatment of the chronic diseasewith which the AI is associated. Unfortunately, such chronic conditionsmay be refractory to treatment, and many patients live with AI as partof their overall health problems. In more severe cases, bloodtransfusions, or commercially-produced erythropoietin, can be helpful insome circumstances, although both treatments are costly, and may bedangerous (see, e.g., Zarychanski R, Houston D. S., Can. Med. Assoc. J.179 (4): 333-7 (2008)). Intravenous infusion of iron has also been usedto treat AI, although the iron compounds that are infused may be potentoxidants which are potentially harmful to the body (see, e.g., R. A.Zager, Clin. J. Am. Soc. Nephrol. 1 Suppl 1: S24-31 (Sep. 2006)).

Thus, there is a continuing need for compositions and methods fortreating, preventing, and/or ameliorating one or more symptoms of AI.

BRIEF SUMMARY OF THE INVENTION

SMAD4 is a 552-amino acid protein involved in cell signaling. SMAD4binds receptor-regulated SMADs (R-SMADs), such as SMAD1 and SMAD2, andfacilitates the translocation of the heteromeric complex into thenucleus where the complex binds to DNA and serves as a transcriptionfactor. For example, SMAD4 modulates the activity of members of the TGFβprotein superfamily, consequently SMAD4 is involved in many cellfunctions, such as differentiation, apoptosis, gastrulation, embryonicdevelopment and the cell cycle.

Wang et al. proposed a role for the SMAD4 protein in the regulation ofiron metabolism. (Wang et al., Cell Metabolism, Vol. 2, pp. 399-409(Dec. 2005)). They showed that liver-specific disruption of the SMAD4gene results in markedly decreased hepcidin expression and a consequentaccumulation of iron in many organs. They postulated a role for SMAD4,acting through modulation of TGFβ, in regulating hepcidin expression andthus iron homeostasis.

Accordingly, it is an object of the present invention to providecompositions and methods for inhibiting the expression of the SMAD4gene. Inhibition is through the mechanism of RNA interference. Thecompositions and methods of the present invention are thus useful, forexample, for treating anemia of inflammation in a human being.

Thus, the present invention provides compositions comprising therapeuticnucleic acids such as interfering RNA (e.g., dsRNA such as siRNA) thattarget SMAD4 gene expression, lipid particles comprising one or more(e.g., a cocktail) of the therapeutic nucleic acids, methods of makingthe lipid particles, and methods of delivering and/or administering thelipid particles (e.g., for treating anemia of inflammation).

More particularly, the invention provides compositions comprisingunmodified and chemically modified interfering RNA (e.g., siRNA)molecules which inhibit or silence SMAD4 gene expression. The presentinvention also provides serum-stable nucleic acid-lipid particles (e.g.,SNALP) and formulations thereof comprising one or more (e.g., acocktail) of the interfering RNA (e.g., siRNA) described herein, acationic lipid, and a non-cationic lipid, which can further comprise aconjugated lipid that inhibits aggregation of particles. Examples ofinterfering RNA molecules include, but are not limited to,double-stranded RNA (dsRNA) such as siRNA, Dicer-substrate dsRNA, shRNA,aiRNA, pre-miRNA, and combinations thereof.

In one aspect, the present invention provides an interfering RNA thattargets SMAD4 gene expression, wherein the interfering RNA comprises asense strand and a complementary antisense strand, and wherein theinterfering RNA comprises a double-stranded region of about 15 to about60 nucleotides in length. In certain embodiments, the present inventionprovides compositions comprising a combination (e.g., a cocktail) of atleast about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more interfering RNAmolecules that target the same and/or different regions of the SMAD4genome. The interfering RNA of the invention are capable of inhibitingor silencing SMAD4 gene expression in vitro and in vivo.

A non-limiting example of a SMAD4 transcript sequence that can be used,for example, in the design of siRNA molecules that inhibit SMAD4 geneexpression is set forth in Genbank (www.ncbi.nlm.nih.gov/genbank) asAccession No. NM_(—)005359.5 (Gene ID: 4089. Official Symbol: SMAD4, andName: SMAD family member 4 [Homo sapiens]).

In another aspect, the present invention provides an interfering RNAthat targets SMAD4 gene expression, wherein the interfering RNAcomprises a sense strand and a complementary antisense strand, andwherein the interfering RNA comprises a double-stranded region of about15 to about 60 nucleotides in length. In certain embodiments, thepresent invention provides compositions comprising a combination (e.g.,a cocktail) of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreinterfering RNA molecules that target the same and/or different regionsof the SMAD4 gene. The interfering RNA of the invention are capable ofinhibiting or completely silencing SMAD4 gene expression in vitro and invivo.

Each of the interfering RNA sequences present in the compositions of theinvention may independently comprise at least one, two, three, four,five, six, seven, eight, nine, ten, or more modified nucleotides such as2′OMe nucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region. Preferably, uridine and/or guanosine nucleotidesare modified with 2′OMe nucleotides. In particular embodiments, each ofthe interfering RNA sequences present in the compositions of theinvention comprises at least one 2′OMe-uridine nucleotide and at leastone 2′OMe-guanosine nucleotide in the sense and/or antisense strands.

The present invention also provides a pharmaceutical compositioncomprising one or a cocktail of interfering RNA (e.g., siRNA) moleculesthat target SMAD4 gene expression, and a pharmaceutically acceptablecarrier.

In another aspect, the present invention provides a nucleic acid-lipidparticle that targets SMAD4 gene expression. The nucleic acid-lipidparticle typically comprises one or more unmodified and/or modifiedinterfering RNA that silence SMAD4 gene expression, a cationic lipid,and a non-cationic lipid. In certain instances, the nucleic acid-lipidparticle further comprises a conjugated lipid that inhibits aggregationof particles. In preferred embodiments, the nucleic acid-lipid particlecomprises one or more unmodified and/or modified interfering RNA thatsilence SMAD4 gene expression, a cationic lipid, a non-cationic lipid,and a conjugated lipid that inhibits aggregation of particles.

In other embodiments, the interfering RNA molecules of the invention arefully encapsulated in the nucleic acid-lipid particle (e.g., SNALP).With respect to formulations comprising a cocktail of interfering RNA,the different types of interfering RNA molecules may be co-encapsulatedin the same nucleic acid-lipid particle, or each type of interfering RNAspecies present in the cocktail may be encapsulated in its own particle.

The present invention also provides pharmaceutical compositionscomprising a nucleic acid-lipid particle and a pharmaceuticallyacceptable carrier.

The nucleic acid-lipid particles of the invention are useful for theprophylactic or therapeutic delivery of interfering RNA (e.g., dsRNA)molecules that silence the expression of one or more SMAD4 genes. Insome embodiments, one or more of the interfering RNA molecules describedherein are formulated into nucleic acid-lipid particles, and theparticles are administered to a mammal (e.g., a human) requiring suchtreatment. In certain instances, a therapeutically effective amount ofthe nucleic acid-lipid particle can be administered to the mammal, e.g.,for preventing or treating anemia of inflammation in a human being).Administration of the nucleic acid-lipid particle can be by any routeknown in the art, such as, e.g., oral, intranasal, intravenous,intraperitoneal, intramuscular, intra-articular, intralesional,intratracheal, subcutaneous, or intradermal. In particular embodiments,the nucleic acid-lipid particle is administered systemically, e.g., viaenteral or parenteral routes of administration.

In some embodiments, downregulation of SMAD4 gene expression isdetermined by detecting SMAD4 RNA or protein levels in a biologicalsample from a mammal after nucleic acid-lipid particle administration.In other embodiments, downregulation of SMAD4 gene expression isdetermined by detecting SMAD4 mRNA or protein levels in a biologicalsample from a mammal after nucleic acid-lipid particle administration.In certain embodiments, downregulation of SMAD4 or SMAD4 gene expressionis detected by monitoring symptoms associated with anemia ofinflammation in a mammal after particle administration.

In another embodiment, the present invention provides methods forintroducing an interfering RNA that silences SMAD4 gene expression intoa cell, the method comprising the step of contacting the cell with anucleic acid-lipid particle of the present invention.

In another embodiment, the present invention provides methods forsilencing SMAD4 gene expression in a mammal (e.g., a human) in needthereof, wherein the methods each include the step of administering tothe mammal a nucleic acid-lipid particle of the present invention.

In another aspect, the present invention provides methods for treatingand/or ameliorating one or more symptoms associated with anemia ofinflammation in a human, wherein the methods each include the step ofadministering to the human a therapeutically effective amount of anucleic acid-lipid particle of the present invention.

In another aspect, the present invention provides methods for inhibitingthe expression of SMAD4 in a mammal in need thereof (e.g., a humansuffering from anemia of inflammation), wherein the methods each includethe step of administering to the mammal a therapeutically effectiveamount of a nucleic acid-lipid particle of the present invention.

In a further aspect, the present invention provides methods forpreventing and/or treating anemia of inflammation in a human, whereinthe methods each include the step of administering to the human atherapeutically effective amount of a nucleic acid-lipid particle of thepresent invention.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and examples.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The interfering RNA (e.g., siRNA) drug therapy described hereinadvantageously provides significant new compositions and methods fortreating anemia of inflammation in human beings. Embodiments of thepresent invention can be administered, for example, once per day, onceper week, or once every several weeks (e.g., once every two, three,four, five or six weeks).

Furthermore, the lipid particles described herein (e.g., SNALP) enablethe effective delivery of a nucleic acid drug such as an interfering RNAinto target tissues and cells within the body. The presence of the lipidparticle confers protection from nuclease degradation in thebloodstream, allows preferential accumulation in target tissue andprovides a means of drug entry into the cellular cytoplasm where thesiRNAs can perform their intended function of RNA interference.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “interfering RNA” or “RNAi” or “interfering RNA sequence” asused herein includes single-stranded RNA (e.g., mature miRNA, ssRNAioligonucleotides, ssDNAi oligonucleotides) or double-stranded RNA (i.e.,duplex RNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, orpre-miRNA) that is capable of reducing or inhibiting the expression of atarget gene or sequence (e.g., by mediating the degradation orinhibiting the translation of mRNAs which are complementary to theinterfering RNA sequence) when the interfering RNA is in the same cellas the target gene or sequence. Interfering RNA thus refers to thesingle-stranded RNA that is complementary to a target mRNA sequence orto the double-stranded RNA formed by two complementary strands or by asingle, self-complementary strand. Interfering RNA may have substantialor complete identity to the target gene or sequence, or may comprise aregion of mismatch (i.e., a mismatch motif). The sequence of theinterfering RNA can correspond to the full-length target gene, or asubsequence thereof. Preferably, the interfering RNA molecules arechemically synthesized.

Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g.,interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides inlength, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotidesin length, and is preferably about 20-24, 21-22, or 21-23 (duplex)nucleotides in length (e.g., each complementary sequence of thedouble-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25nucleotides in length, preferably about 20-24, 21-22, or 21-23nucleotides in length, and the double-stranded siRNA is about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferablyabout 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes maycomprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 toabout 3 nucleotides and 5′ phosphate termini. Examples of siRNA include,without limitation, a double-stranded polynucleotide molecule assembledfrom two separate stranded molecules, wherein one strand is the sensestrand and the other is the complementary antisense strand; adouble-stranded polynucleotide molecule assembled from a single strandedmolecule, where the sense and antisense regions are linked by a nucleicacid-based or non-nucleic acid-based linker; a double-strandedpolynucleotide molecule with a hairpin secondary structure havingself-complementary sense and antisense regions; and a circularsingle-stranded polynucleotide molecule with two or more loop structuresand a stem having self-complementary sense and antisense regions, wherethe circular polynucleotide can be processed in vivo or in vitro togenerate an active double-stranded siRNA molecule.

Preferably, siRNA are chemically synthesized. siRNA can also begenerated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25nucleotides in length) with the E. coli RNase III or Dicer. Theseenzymes process the dsRNA into biologically active siRNA (see, e.g.,Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegariet al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al.,Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res.,31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); andRobertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA areat least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotidesin length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotidesin length, or longer. The dsRNA can encode for an entire gene transcriptor a partial gene transcript. In certain instances, siRNA may be encodedby a plasmid (e.g., transcribed as sequences that automatically foldinto duplexes with hairpin loops).

As used herein, the term “mismatch motif” or “mismatch region” refers toa portion of an interfering RNA (e.g., siRNA) sequence that does nothave 100% complementarity to its target sequence. An interfering RNA mayhave at least one, two, three, four, five, six, or more mismatchregions. The mismatch regions may be contiguous or may be separated by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatchmotifs or regions may comprise a single nucleotide or may comprise two,three, four, five, or more nucleotides.

The phrase “inhibiting expression of a target gene” refers to theability of an interfering RNA (e.g., siRNA) of the invention to silence,reduce, or inhibit expression of a target gene (e.g., SMAD4 gene). Toexamine the extent of gene silencing, a test sample (e.g., a biologicalsample from an organism of interest expressing the target gene or asample of cells in culture expressing the target gene) is contacted withan interfering RNA (e.g., siRNA) that silences, reduces, or inhibitsexpression of the target gene. Expression of the target gene in the testsample is compared to expression of the target gene in a control sample(e.g., a biological sample from an organism of interest expressing thetarget gene or a sample of cells in culture expressing the target gene)that is not contacted with the interfering RNA (e.g., siRNA). Controlsamples (e.g., samples expressing the target gene) may be assigned avalue of 100%. In particular embodiments, silencing, inhibition, orreduction of expression of a target gene is achieved when the value ofthe test sample relative to the control sample is about 95%, 90%, 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%,5%, or 0%. Suitable assays include, without limitation, examination ofprotein or mRNA levels using techniques known to those of skill in theart, such as, e.g., dot blots, Northern blots, in situ hybridization,ELISA, immunoprecipitation, enzyme function, as well as phenotypicassays known to those of skill in the art.

An “effective amount” or “therapeutically effective amount” of atherapeutic nucleic acid such as an interfering RNA is an amountsufficient to produce the desired effect, e.g., an inhibition ofexpression of a target sequence in comparison to the normal expressionlevel detected in the absence of an interfering RNA. In particularembodiments, inhibition of expression of a target gene or targetsequence is achieved when the value obtained with an interfering RNArelative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitableassays for measuring the expression of a target gene or target sequenceinclude, but are not limited to, examination of protein or mRNA levelsusing techniques known to those of skill in the art, such as, e.g., dotblots, Northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, as well as phenotypic assays knownto those of skill in the art.

By “decrease,” “decreasing,” “reduce,” or “reducing” of an immuneresponse by an interfering RNA is intended to mean a detectable decreaseof an immune response to a given interfering RNA (e.g., a modifiedinterfering RNA). The amount of decrease of an immune response by amodified interfering RNA may be determined relative to the level of animmune response in the presence of an unmodified interfering RNA. Adetectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or morelower than the immune response detected in the presence of theunmodified interfering RNA. A decrease in the immune response tointerfering RNA is typically measured by a decrease in cytokineproduction (e.g., IFNγ, IFNα, TNFα, IL-6, or IL-12) by a responder cellin vitro or a decrease in cytokine production in the sera of a mammaliansubject after administration of the interfering RNA.

As used herein, the term “responder cell” refers to a cell, preferably amammalian cell, that produces a detectable immune response whencontacted with an immunostimulatory interfering RNA such as anunmodified siRNA. Exemplary responder cells include, e.g., dendriticcells, macrophages, peripheral blood mononuclear cells (PBMCs),splenocytes, and the like. Detectable immune responses include, e.g.,production of cytokines or growth factors such as TNF-α, IFN-α, IFN-β,IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TGF, andcombinations thereof. Detectable immune responses also include, e.g.,induction of interferon-induced protein with tetratricopeptide repeats 1(IFIT1) mRNA.

“Substantial identity” refers to a sequence that hybridizes to areference sequence under stringent conditions, or to a sequence that hasa specified percent identity over a specified region of a referencesequence.

The phrase “stringent hybridization conditions” refers to conditionsunder which a nucleic acid will hybridize to its target sequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T^(m)) for thespecific sequence at a defined ionic strength pH. The T^(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T^(m), 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization.

Exemplary stringent hybridization conditions can be as follows: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec. to 2 min., an annealingphase lasting 30 sec. to 2 min., and an extension phase of about 72° C.for 1 to 2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y. (1990).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous references, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds.

The terms “substantially identical” or “substantial identity,” in thecontext of two or more nucleic acids, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same (i.e., at least about 60%, preferably atleast about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over aspecified region), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. This definition, when the context indicates, alsorefers analogously to the complement of a sequence. Preferably, thesubstantial identity exists over a region that is at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of a number of contiguous positions selected from the groupconsisting of from about 5 to about 60, usually about 10 to about 45,more usually about 15 to about 30, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.,48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESSMAD4IT, FASTA, andSMAD4ASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Current Protocols in Molecular Biology,Ausubel et al., eds. (1995 supplement)).

Non-limiting examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.,215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). Anotherexample is a global alignment algorithm for determining percent sequenceidentity such as the Needleman-Wunsch algorithm for aligning protein ornucleotide (e.g., RNA) sequences.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA and RNA. DNA may be in the formof, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCRproduct, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expressioncassettes, chimeric sequences, chromosomal DNA, or derivatives andcombinations of these groups. RNA may be in the form of smallinterfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA(shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA,tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleicacids include nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, and which have similar bindingproperties as the reference nucleic acid. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2′-O-methyl ribonucleotides,and peptide-nucleic acids (PNAs). Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid. Unless otherwise indicated, a particular nucleic acidsequence also implicitly encompasses conservatively modified variantsthereof (e.g., degenerate codon substitutions), alleles, orthologs,SNPs, and complementary sequences as well as the sequence explicitlyindicated. Specifically, degenerate codon substitutions may be achievedby generating sequences in which the third position of one or moreselected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991);Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes, 8:91-98 (1994)). “Nucleotides” contain a sugardeoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.Nucleotides are linked together through the phosphate groups. “Bases”include purines and pyrimidines, which further include natural compoundsadenine, thymine, guanine, cytosine, uracil, inosine, and naturalanalogs, and synthetic derivatives of purines and pyrimidines, whichinclude, but are not limited to, modifications which place new reactivegroups such as, but not limited to, amines, alcohols, thiols,carboxylates, and alkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids; and (3) “derived lipids” such assteroids.

The term “lipid particle” includes a lipid formulation that can be usedto deliver a therapeutic nucleic acid (e.g., interfering RNA) to atarget site of interest (e.g., cell, tissue, organ, and the like). Inpreferred embodiments, the lipid particle of the invention is a nucleicacid-lipid particle, which is typically formed from a cationic lipid, anon-cationic lipid, and optionally a conjugated lipid that preventsaggregation of the particle. In other preferred embodiments, thetherapeutic nucleic acid (e.g., interfering RNA) may be encapsulated inthe lipid portion of the particle, thereby protecting it from enzymaticdegradation.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a particle made from lipids (e.g., acationic lipid, a non-cationic lipid, and optionally a conjugated lipidthat prevents aggregation of the particle), wherein the nucleic acid(e.g., interfering RNA) is fully encapsulated within the lipid. Incertain instances, SNALP are extremely useful for systemic applications,as they can exhibit extended circulation lifetimes following intravenous(i.v.) injection, they can accumulate at distal sites (e.g., sitesphysically separated from the administration site), and they can mediatesilencing of target gene expression at these distal sites. The nucleicacid may be complexed with a condensing agent and encapsulated within aSNALP as set forth in PCT Publication No. WO 00/03683, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes.

The lipid particles of the invention (e.g., SNALP) typically have a meandiameter of from about 30 nm to about 150 nm, from about 40 nm to about150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm,from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In addition, nucleic acids, whenpresent in the lipid particles of the present invention, are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Patent Publication Nos. 20040142025 and 20070042031, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

As used herein, “lipid encapsulated” can refer to a lipid particle thatprovides a therapeutic nucleic acid such as an interfering RNA (e.g.,siRNA), with full encapsulation, partial encapsulation, or both. In apreferred embodiment, the nucleic acid (e.g., interfering RNA) is fullyencapsulated in the lipid particle (e.g., to form a SNALP or othernucleic acid-lipid particle).

The term “lipid conjugate” refers to a conjugated lipid that inhibitsaggregation of lipid particles. Such lipid conjugates include, but arenot limited to, PEG-lipid conjugates such as, e.g., PEG coupled todialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled todiacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol,PEG coupled to phosphatidylethanolamines, and PEG conjugated toceramides (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids,polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates; see,e.g., U.S. Provisional Application No. 61/294,828, filed Jan. 13, 2010,and U.S. Provisional Application No. 61/295, 140, filed Jan. 14, 2010),polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof.Additional examples of POZ-lipid conjugates are described in PCTPublication No. WO 2010/006282. PEG or POZ can be conjugated directly tothe lipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG or the POZ to a lipid can be usedincluding, e.g., non-ester containing linker moieties andester-containing linker moieties. In certain preferred embodiments,non-ester containing linker moieties, such as amides or carbamates, areused. The disclosures of each of the above patent documents are hereinincorporated by reference in their entirety for all purposes.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long-chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic, or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids, and sphingolipids.

Representative examples of phospholipids include, but are not limitedto, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, are also within the group designated as amphipathiclipids. Additionally, the amphipathic lipids described above can bemixed with other lipids including triglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The terms “cationic lipid” and “amino lipid” are used interchangeablyherein to include those lipids and salts thereof having one, two, three,or more fatty acid or fatty alkyl chains and a pH-titratable amino headgroup (e.g., an alkylamino or dialkylamino head group). The cationiclipid is typically protonated (i.e., positively charged) at a pH belowthe pK_(a) of the cationic lipid and is substantially neutral at a pHabove the pK_(a). The cationic lipids of the invention may also betermed titratable cationic lipids. In some embodiments, the cationiclipids comprise: a protonatable tertiary amine (e.g., pH-titratable)head group; C₁₈ alkyl chains, wherein each alkyl chain independently has0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketallinkages between the head group and alkyl chains. Such cationic lipidsinclude, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA,γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2,and C2K), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, γ-DLen-C2K-DMA,DLin-M-C2-DMA (also known as MC2), and DLin-M-C3-DMA (also known asMC3).

The term “salts” includes any anionic and cationic complex, such as thecomplex formed between a cationic lipid and one or more anions.Non-limiting examples of anions include inorganic and organic anions,e.g., hydride, fluoride, chloride, bromide, iodide, oxalate (e.g.,hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogenphosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride,bisulfate, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogensulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate,acrylate, polyacrylate, fumarate, maleate, itaconate, glycolate,gluconate, malate, mandelate, tiglate, ascorbate, salicylate,polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite,bromate, hypobromite, iodate, an alkylsulfonate, an arylsulfonate,arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate,hydroxide, peroxide, permanganate, and mixtures thereof. In particularembodiments, the salts of the cationic lipids disclosed herein arecrystalline salts.

The term “alkyl” includes a straight chain or branched, noncyclic orcyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbonatoms. Representative saturated straight chain alkyls include, but arenot limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, andthe like, while saturated branched alkyls include, without limitation,isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Representative saturated cyclic alkyls include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, whileunsaturated cyclic alkyls include, without limitation, cyclopentenyl,cyclohexenyl, and the like.

The term “alkenyl” includes an alkyl, as defined above, containing atleast one double bond between adjacent carbon atoms. Alkenyls includeboth cis and trans isomers. Representative straight chain and branchedalkenyls include, but are not limited to, ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike.

The term “alkynyl” includes any alkyl or alkenyl, as defined above,which additionally contains at least one triple bond between adjacentcarbons. Representative straight chain and branched alkynyls include,without limitation, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

The term “acyl” includes any alkyl, alkenyl, or alkynyl wherein thecarbon at the point of attachment is substituted with an oxo group, asdefined below. The following are non-limiting examples of acyl groups:—C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl.

The term “heterocycle” includes a 5- to 7-membered monocyclic, or 7- to10-membered bicyclic, heterocyclic ring which is either saturated,unsaturated, or aromatic, and which contains from 1 or 2 heteroatomsindependently selected from nitrogen, oxygen and sulfur, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen heteroatom may be optionally quaternized, including bicyclicrings in which any of the above heterocycles are fused to a benzenering. The heterocycle may be attached via any heteroatom or carbon atom.Heterocycles include, but are not limited to, heteroaryls as definedbelow, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl,oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

The term “heterocycle” includes a 5- to 7-membered monocyclic, or 7- to10-membered bicyclic, heterocyclic ring which is either saturated,unsaturated, or aromatic, and which contains from 1 or 2 heteroatomsindependently selected from nitrogen, oxygen and sulfur, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen heteroatom may be optionally quaternized, including bicyclicrings in which any of the above heterocycles are fused to a benzenering. The heterocycle may be attached via any heteroatom or carbon atom.Heterocycles include, but are not limited to, heteroaryls as definedbelow, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl,oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” mean that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O), two hydrogen atoms are replaced.In this regard, substituents include, but are not limited to, oxo,halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y),—NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y),—SO_(n)R^(x), and —SO_(n)NR^(x)R^(y), wherein n is 0, 1, or 2, R^(x) andR^(y) are the same or different and are independently hydrogen, alkyl,or heterocycle, and each of the alkyl and heterocycle substituents maybe further substituted with one or more of oxo, halogen, —OH, —CN,alkyl, —OR^(x), heterocycle, —NR^(x)R^(y), —NR^(x)C(═O)R^(y),—NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y),—SO_(n)R^(x), and —SO_(n)NR^(x)R^(y). The term “optionally substituted,”when used before a list of substituents, means that each of thesubstituents in the list may be optionally substituted as describedherein.

The term “halogen” includes fluoro, chloro, bromo, and iodo.

The term “fusogenic” refers to the ability of a lipid particle, such asa SNALP, to fuse with the membranes of a cell. The membranes can beeither the plasma membrane or membranes surrounding organelles, e.g.,endosome, nucleus, etc.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

“Distal site,” as used herein, refers to a physically separated site,which is not limited to an adjacent capillary bed, but includes sitesbroadly distributed throughout an organism.

“Serum-stable” in relation to nucleic acid-lipid particles such as SNALPmeans that the particle is not significantly degraded after exposure toa serum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of lipidparticles that leads to a broad biodistribution of an active agent suchas an interfering RNA (e.g., siRNA) within an organism. Some techniquesof administration can lead to the systemic delivery of certain agents,but not others. Systemic delivery means that a useful, preferablytherapeutic, amount of an agent is exposed to most parts of the body. Toobtain broad biodistribution generally requires a blood lifetime suchthat the agent is not rapidly degraded or cleared (such as by first passorgans (liver, lung, etc.) or by rapid, nonspecific cell binding) beforereaching a disease site distal to the site of administration. Systemicdelivery of lipid particles can be by any means known in the artincluding, for example, intravenous, subcutaneous, and intraperitoneal.In a preferred embodiment, systemic delivery of lipid particles is byintravenous delivery.

“Local delivery,” as used herein, refers to delivery of an active agentsuch as an interfering RNA (e.g., siRNA) directly to a target sitewithin an organism. For example, an agent can be locally delivered bydirect injection into a disease site, other target site, or a targetorgan such as the liver, heart, pancreas, kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

The term “reticuloendothelial system” or “RES” refers to the part of theimmune system that contains reticuloendothelial cells, including thephagocytic cells located in reticular connective tissue such asmonocytes and macrophages. These cells typically accumulate in lymphnodes and the spleen. The Kupffer cells of the liver and tissuehistiocytes are also part of the RES. The RES is divided into primaryand secondary lymphoid organs. Primary (“central”) lymphoid organs arethe sites where the cells of the RES are produced. The cells of the RESare produced in the bone marrow. The thymus is also included as it isthe required site for T cell maturation. Secondary (“peripheral”)lymphoid organs are the sites where the cells of the RES function. Thisincludes the lymph nodes, tonsils, spleen, and “MALT” (mucosa-associatedlymphoid tissue). MALT is further divided into “GALT” (gut-associatedlymphoid tissue) and “BALT” (bronchus-associated lymphoid tissue). TheKupffer cells of the liver act as part of this system, but are notorganized into a tissue; rather, they are dispersed throughout the liversinusoids. The microglia of the central nervous system (CNS) can beconsidered a part of the RES. They are scavenger cells that proliferatein response to CNS injury.

III. Description of the Embodiments

The present invention provides therapeutic nucleic acids such asinterfering RNA (e.g., dsRNA such as siRNA) that target the expressionof SMAD4 genes, lipid particles comprising one or more (e.g., acocktail) of the therapeutic nucleic acids, methods of making the lipidparticles, and methods of delivering and/or administering the lipidparticles (e.g., for the treatment of anemia of inflammation in humans).

In one aspect, the present invention provides interfering RNA moleculesthat target SMAD4 gene expression. Non-limiting examples of interferingRNA molecules include double-stranded RNA capable of mediating RNAi suchas siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, pre-miRNA, and mixturesthereof. In certain instances, the present invention providescompositions comprising a combination (e.g., a cocktail, pool, ormixture) of interfering RNAs that target different regions of the SMAD4gene. In certain instances, the interfering RNA (e.g., siRNA) moleculesof the invention are capable of silencing SMAD4 gene expression,inactivating SMAD4, and/or inhibiting the replication of SMAD4 in vitroor in vivo.

In particular embodiments, the present invention provides an interferingRNA (e.g., siRNA) that silences SMAD4 gene expression, wherein theinterfering RNA comprises a sense strand and a complementary antisensestrand, and wherein the interfering RNA comprises a double-strandedregion of about 15 to about 60 nucleotides in length (e.g., about 15-60,15-30, 15-25, 19-30, 19-25, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35,20-30, 20-25, 21-30, 21-29, 22-30, 22-29, 22-28, 23-30, 23-28, 24-30,24-28, 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides inlength, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, or 35 nucleotides in length).

In certain embodiments, the interfering RNA (e.g., siRNA) of the presentinvention may comprise at least one, two, three, four, five, six, seven,eight, nine, ten, or more modified nucleotides such as 2′OMenucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region of the interfering RNA. Preferably, uridineand/or guanosine nucleotides in the interfering RNA are modified with2′OMe nucleotides. In certain instances, the interfering RNA contains2′OMe nucleotides in both the sense and antisense strands and comprisesat least one 2′OMe-uridine nucleotide and at least one 2′OMe-guanosinenucleotide in the double-stranded region. In some embodiments, the senseand/or antisense strand of the interfering RNA may further comprisemodified (e.g., 2′OMe-modified) adenosine and/or modified (e.g.,2′OMe-modified) cytosine nucleotides, e.g., in the double-strandedregion of the interfering RNA.

In particular embodiments, the interfering RNA (e.g., siRNA) moleculesof the present invention comprise a 3′ overhang of 1, 2, 3, or 4nucleotides in one or both strands. In certain instances, theinterfering RNA may contain at least one blunt end. In particularembodiments, the 3′ overhangs in one or both strands of the interferingRNA may each independently comprise 1, 2, 3, or 4 modified and/orunmodified deoxythymidine (“t” or “dT”) nucleotides, 1, 2, 3, or 4modified (e.g., 2′OMe) and/or unmodified uridine (“U”) ribonucleotides,or 1, 2, 3, or 4 modified (e.g., 2′OMe) and/or unmodifiedribonucleotides or deoxyribonucleotides having complementarity to thetarget sequence or the complementary strand thereof.

The present invention also provides a pharmaceutical compositioncomprising one or more (e.g., a cocktail) of the interfering RNAsdescribed herein and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a nucleic acid-lipidparticle (e.g., SNALP) that targets SMAD4 gene expression. The nucleicacid-lipid particles (e.g., SNALP) typically comprise one or more (e.g.,a cocktail) of the interfering RNAs described herein, a cationic lipid,and a non-cationic lipid. In certain instances, the nucleic acid-lipidparticles (e.g., SNALP) further comprise a conjugated lipid thatinhibits aggregation of particles. Preferably, the nucleic acid-lipidparticles (e.g., SNALP) comprise one or more (e.g., a cocktail) of theinterfering RNAs described herein, a cationic lipid, a non-cationiclipid, and a conjugated lipid that inhibits aggregation of particles.

In some embodiments, the interfering RNAs (e.g., siRNAs) of the presentinvention are fully encapsulated in the nucleic acid-lipid particle(e.g., SNALP). With respect to formulations comprising an interferingRNA cocktail, the different types of interfering RNA species present inthe cocktail (e.g., interfering RNA compounds with different sequences)may be co-encapsulated in the same particle, or each type of interferingRNA species present in the cocktail may be encapsulated in a separateparticle. The interfering RNA cocktail may be formulated in theparticles described herein using a mixture of two or more individualinterfering RNAs (each having a unique sequence) at identical, similar,or different concentrations or molar ratios. In one embodiment, acocktail of interfering RNAs (corresponding to a plurality ofinterfering RNAs with different sequences) is formulated usingidentical, similar, or different concentrations or molar ratios of eachinterfering RNA species, and the different types of interfering RNAs areco-encapsulated in the same particle. In another embodiment, each typeof interfering RNA species present in the cocktail is encapsulated indifferent particles at identical, similar, or different interfering RNAconcentrations or molar ratios, and the particles thus formed (eachcontaining a different interfering RNA payload) are administeredseparately (e.g., at different times in accordance with a therapeuticregimen), or are combined and administered together as a single unitdose (e.g., with a pharmaceutically acceptable carrier). The particlesdescribed herein are serum-stable, are resistant to nucleasedegradation, and are substantially non-toxic to mammals such as humans.

The cationic lipid in the nucleic acid-lipid particles of the invention(e.g., SNALP) may comprise, e.g., one or more cationic lipids of FormulaI-III described herein or any other cationic lipid species. In oneparticular embodiment, the cationic lipid is selected from the groupconsisting of 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin-M-C3-DMA), salts thereof, and mixturesthereof.

The non-cationic lipid in the nucleic acid-lipid particles of thepresent invention (e.g., SNALP) may comprise, e.g., one or more anioniclipids and/or neutral lipids. In some embodiments, the non-cationiclipid comprises one of the following neutral lipid components: (1) amixture of a phospholipid and cholesterol or a derivative thereof; (2)cholesterol or a derivative thereof; or (3) a phospholipid. In certainpreferred embodiments, the phospholipid comprisesdipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), or a mixture thereof. In a particularly preferred embodiment,the non-cationic lipid is a mixture of DPPC and cholesterol.

The lipid conjugate in the nucleic acid-lipid particles of the invention(e.g., SNALP) inhibits aggregation of particles and may comprise, e.g.,one or more of the lipid conjugates described herein. In one particularembodiment, the lipid conjugate comprises a PEG-lipid conjugate.Examples of PEG-lipid conjugates include, but are not limited to,PEG-DAG conjugates, PEG-DAA conjugates, and mixtures thereof. In certainembodiments, the PEG-DAA conjugate in the lipid particle may comprise aPEG-didecyloxypropyl (C10) conjugate, a PEG-dilauryloxypropyl (C12)conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, aPEG-dipalmityloxypropyl (C16) conjugate, a PEG-distearyloxypropyl (C₁₈)conjugate, or mixtures thereof. In another embodiment, the lipidconjugate comprises a POZ-lipid conjugate such as a POZ-DAA conjugate.

In some embodiments, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) comprising: (a) one or more (e.g., a cocktail)interfering RNA molecules that target SMAD4 gene expression; (b) one ormore cationic lipids or salts thereof comprising from about 50 mol % toabout 85 mol % of the total lipid present in the particle; (c) one ormore non-cationic lipids comprising from about 13 mol % to about 49.5mol % of the total lipid present in the particle; and (d) one or moreconjugated lipids that inhibit aggregation of particles comprising fromabout 0.5 mol % to about 2 mol % of the total lipid present in theparticle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) one or more (e.g., a cocktail) interfering RNA moleculesthat target SMAD4 gene expression; (b) a cationic lipid or a saltthereof comprising from about 52 mol % to about 62 mol % of the totallipid present in the particle; (c) a mixture of a phospholipid andcholesterol or a derivative thereof comprising from about 36 mol % toabout 47 mol % of the total lipid present in the particle; and (d) aPEG-lipid conjugate comprising from about 1 mol % to about 2 mol % ofthe total lipid present in the particle. This embodiment of nucleicacid-lipid particle is generally referred to herein as the “1:57”formulation. In one particular embodiment, the 1:57 formulation is afour-component system comprising about 1.4 mol % PEG-lipid conjugate(e.g., PEG2000-C-DMA), about 57.1 mol % cationic lipid (e.g.,DLin-K-C2-DMA) or a salt thereof, about 7.1 mol % DPPC (or DSPC), andabout 34.3 mol % cholesterol (or derivative thereof).

In another aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) one or more (e.g., a cocktail) interfering RNA moleculesthat target SMAD4 gene expression; (b) a cationic lipid or a saltthereof comprising from about 56.5 mol % to about 66.5 mol % of thetotal lipid present in the particle; (c) cholesterol or a derivativethereof comprising from about 31.5 mol % to about 42.5 mol % of thetotal lipid present in the particle; and (d) a PEG-lipid conjugatecomprising from about 1 mol % to about 2 mol % of the total lipidpresent in the particle. This embodiment of nucleic acid-lipid particleis generally referred to herein as the “1:62” formulation. In oneparticular embodiment, the 1:62 formulation is a three-component systemwhich is phospholipid-free and comprises about 1.5 mol % PEG-lipidconjugate (e.g., PEG2000-C-DMA), about 61.5 mol % cationic lipid (e.g.,DLin-K-C2-DMA) or a salt thereof, and about 36.9 mol % cholesterol (orderivative thereof).

Additional embodiments related to the 1:57 and 1:62 formulations aredescribed in PCT Publication No. WO 09/127,060 and published US patentapplication publication number US 2011/0071208 A1, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

In other embodiments, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) comprising: (a) one or more (e.g., a cocktail)interfering RNA molecules that target SMAD4 gene expression; (b) one ormore cationic lipids or salts thereof comprising from about 2 mol % toabout 50 mol % of the total lipid present in the particle; (c) one ormore non-cationic lipids comprising from about 5 mol % to about 90 mol %of the total lipid present in the particle; and (d) one or moreconjugated lipids that inhibit aggregation of particles comprising fromabout 0.5 mol % to about 20 mol % of the total lipid present in theparticle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) one or more (e.g., a cocktail) interfering RNA moleculesthat target SMAD4 gene expression; (b) a cationic lipid or a saltthereof comprising from about 30 mol % to about 50 mol % of the totallipid present in the particle; (c) a mixture of a phospholipid andcholesterol or a derivative thereof comprising from about 47 mol % toabout 69 mol % of the total lipid present in the particle; and (d) aPEG-lipid conjugate comprising from about 1 mol % to about 3 mol % ofthe total lipid present in the particle. This embodiment of nucleicacid-lipid particle is generally referred to herein as the “2:40”formulation. In one particular embodiment, the 2:40 formulation is afour-component system which comprises about 2 mol % PEG-lipid conjugate(e.g., PEG2000-C-DMA), about 40 mol % cationic lipid (e.g.,DLin-K-C2-DMA) or a salt thereof, about 10 mol % DPPC (or DSPC), andabout 48 mol % cholesterol (or derivative thereof).

In further embodiments, the present invention provides nucleicacid-lipid particles (e.g., SNALP) comprising: (a) one or more (e.g., acocktail) interfering RNA molecules that target SMAD4 gene expression;(b) one or more cationic lipids or salts thereof comprising from about50 mol % to about 65 mol % of the total lipid present in the particle;(c) one or more non-cationic lipids comprising from about 25 mol % toabout 45 mol % of the total lipid present in the particle; and (d) oneor more conjugated lipids that inhibit aggregation of particlescomprising from about 5 mol % to about 10 mol % of the total lipidpresent in the particle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) one or more (e.g., a cocktail) interfering RNA moleculesthat target SMAD4 gene expression; (b) a cationic lipid or a saltthereof comprising from about 50 mol % to about 60 mol % of the totallipid present in the particle; (c) a mixture of a phospholipid andcholesterol or a derivative thereof comprising from about 35 mol % toabout 45 mol % of the total lipid present in the particle; and (d) aPEG-lipid conjugate comprising from about 5 mol % to about 10 mol % ofthe total lipid present in the particle. This embodiment of nucleicacid-lipid particle is generally referred to herein as the “7:54”formulation. In certain instances, the non-cationic lipid mixture in the7:54 formulation comprises: (i) a phospholipid of from about 5 mol % toabout 10 mol % of the total lipid present in the particle; and (ii)cholesterol or a derivative thereof of from about 25 mol % to about 35mol % of the total lipid present in the particle. In one particularembodiment, the 7:54 formulation is a four-component system whichcomprises about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about54 mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 7mol % DPPC (or DSPC), and about 32 mol % cholesterol (or derivativethereof).

In another aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) one or more (e.g., a cocktail) interfering RNA moleculesthat target SMAD4 gene expression; (b) a cationic lipid or a saltthereof comprising from about 55 mol % to about 65 mol % of the totallipid present in the particle; (c) cholesterol or a derivative thereofcomprising from about 30 mol % to about 40 mol % of the total lipidpresent in the particle; and (d) a PEG-lipid conjugate comprising fromabout 5 mol % to about 10 mol % of the total lipid present in theparticle. This embodiment of nucleic acid-lipid particle is generallyreferred to herein as the “7:58” formulation. In one particularembodiment, the 7:58 formulation is a three-component system which isphospholipid-free and comprises about 7 mol % PEG-lipid conjugate (e.g.,PEG750-C-DMA), about 58 mol % cationic lipid (e.g., DLin-K-C2-DMA) or asalt thereof, and about 35 mol % cholesterol (or derivative thereof).

Additional embodiments related to the 7:54 and 7:58 formulations aredescribed in published US patent application publication number US2011/0076335 A1, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

The present invention also provides pharmaceutical compositionscomprising a nucleic acid-lipid particle such as a SNALP and apharmaceutically acceptable carrier.

The nucleic acid-lipid particles of the present invention (e.g., SNALP)are useful for the therapeutic delivery of interfering RNAs (e.g.,siRNAs) that silence the expression of one or more SMAD4 genes. In someembodiments, a cocktail of interfering RNAs that target differentregions (e.g., overlapping and/or non-overlapping sequences) of an SMAD4gene is formulated into the same or different nucleic acid-lipidparticles, and the particles are administered to a mammal (e.g., ahuman) requiring such treatment. In certain instances, a therapeuticallyeffective amount of the nucleic acid-lipid particles can be administeredto the mammal, e.g., for treating anemia of inflammation in a human.

In certain embodiments, the present invention provides a method forintroducing one or more interfering RNA (e.g., siRNA) moleculesdescribed herein into a cell by contacting the cell with a nucleicacid-lipid particle described herein (e.g., a SNALP formulation). In oneparticular embodiment, the cell is a reticuloendothelial cell (e.g.,monocyte or macrophage), fibroblast cell, endothelial cell, or plateletcell.

In some embodiments, the nucleic acid-lipid particles described herein(e.g., SNALP) are administered by one of the following routes ofadministration: oral, intranasal, intravenous, intraperitoneal,intramuscular, intra-articular, intralesional, intratracheal,subcutaneous, and intradermal. In particular embodiments, the nucleicacid-lipid particles are administered systemically, e.g., via enteral orparenteral routes of administration.

In particular embodiments, the nucleic acid-lipid particles of theinvention (e.g., SNALP) can preferentially deliver a payload such as aninterfering RNA (e.g., dsRNA) to the liver as compared to other tissues,e.g., for the treatment of acute or chronic anemia of inflammation.

In certain aspects, the present invention provides methods for silencingSMAD4 gene expression in a mammal (e.g., human) in need thereof, themethod comprising administering to the mammal a therapeuticallyeffective amount of a nucleic acid-lipid particle (e.g., a SNALPformulation) comprising one or more interfering RNAs (e.g., siRNAs)described herein (e.g., siRNAs targeting one or more SMAD4 genes). Insome embodiments, administration of nucleic acid-lipid particlescomprising one or more SMAD4 interfering RNAs reduces SMAD4 RNA levelsby at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range therein) relative toSMAD4 RNA levels detected in the absence of the interfering RNA (e.g.,buffer control or irrelevant non-SMAD4 targeting interfering RNAcontrol). In other embodiments, administration of nucleic acid-lipidparticles comprising one or more SMAD4-targeting interfering RNAsreduces SMAD4 RNA levels for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days or more (orany range therein) relative to a negative control such as, e.g., abuffer control or an irrelevant non-SMAD4 targeting interfering RNAcontrol.

In other aspects, the present invention provides methods for silencingSMAD4 gene expression in a mammal (e.g., human) in need thereof, themethod comprising administering to the mammal a therapeuticallyeffective amount of a nucleic acid-lipid particle (e.g., a SNALPformulation) comprising one or more interfering RNAs (e.g., siRNAs)described herein (e.g., siRNAs targeting one or more regions of theSMAD4 gene). In some embodiments, administration of nucleic acid-lipidparticles comprising one or more SMAD4 interfering RNAs reduces SMAD4mRNA levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any rangetherein) relative to SMAD4 mRNA levels detected in the absence of theinterfering RNA (e.g., buffer control or irrelevant non-SMAD4 targetinginterfering RNA control). In other embodiments, administration ofnucleic acid-lipid particles comprising one or more SMAD4-targetinginterfering RNAs reduces SMAD4 mRNA levels for at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100 days or more (or any range therein) relative to a negative controlsuch as, e.g., a buffer control or an irrelevant non-SMAD4 targetinginterfering RNA control.

In other aspects, the present invention provides methods for treating,preventing, reducing the risk or likelihood of developing (e.g.,reducing the susceptibility to), delaying the onset of, and/orameliorating one or more symptoms associated with anemia of inflammationin a mammal (e.g., human) in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of anucleic acid-lipid particle (e.g., a SNALP formulation) comprising oneor more interfering RNA molecules (e.g., siRNAs) described herein thattarget SMAD4 gene expression.

In further aspects, the present invention provides a method forinactivating SMAD4 in a mammal (e.g., human) in need thereof (e.g., ahuman suffering from anemia of inflammation), the method comprisingadministering to the mammal a therapeutically effective amount of anucleic acid-lipid particle (e.g., a SNALP formulation) comprising oneor more interfering RNAs (e.g., siRNAs) described herein that targetSMAD4 gene expression. In some embodiments, administration of nucleicacid-lipid particles comprising one or more SMAD4-targeting interferingRNAs lowers, reduces, or decreases SMAD4 enzyme levels by at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% (or any range therein) relative to the SMAD4enzyme levels detected in the absence of the interfering RNA (e.g.,buffer control or irrelevant non-SMAD4 targeting interfering RNAcontrol).

By way of example, SMAD4 mRNA can be measured using a branched DNA assay(QuantiGene®; Affymetrix). The branched DNA assay is a sandwich nucleicacid hybridization method that uses bDNA molecules to amplify signalfrom captured target RNA.

IV. Therapeutic Nucleic Acids

The term “nucleic acid” includes any oligonucleotide or polynucleotide,with fragments containing up to 60 nucleotides generally termedoligonucleotides, and longer fragments termed polynucleotides. Inparticular embodiments, oligonucletoides of the invention are from about15 to about 60 nucleotides in length. In some embodiments, nucleic acidis associated with a carrier system such as the lipid particlesdescribed herein. In certain embodiments, the nucleic acid is fullyencapsulated in the lipid particle. Nucleic acid may be administeredalone in the lipid particles of the invention, or in combination (e.g.,co-administered) with lipid particles comprising peptides, polypeptides,or small molecules such as conventional drugs.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally-occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also include polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake, reduced immunogenicity, and increasedstability in the presence of nucleases.

Oligonucleotides are generally classified as deoxyribooligonucleotidesor ribooligonucleotides. A deoxyribooligonucleotide consists of a5-carbon sugar called deoxyribose joined covalently to phosphate at the5′ and 3′ carbons of this sugar to form an alternating, unbranchedpolymer. A ribooligonucleotide consists of a similar repeating structurewhere the 5-carbon sugar is ribose.

The nucleic acid can be single-stranded DNA or RNA, or double-strandedDNA or RNA, or DNA-RNA hybrids. In preferred embodiments, the nucleicacid is double-stranded RNA. Examples of double-stranded RNA aredescribed herein and include, e.g., siRNA and other RNAi agents such asDicer-substrate dsRNA, shRNA, aiRNA, and pre-miRNA. In otherembodiments, the nucleic acid is single-stranded. Single-strandednucleic acids include, e.g., antisense oligonucleotides, ribozymes,mature miRNA, and triplex-forming oligonucleotides.

Nucleic acids of the invention may be of various lengths, generallydependent upon the particular form of nucleic acid. For example, inparticular embodiments, plasmids or genes may be from about 1,000 toabout 100,000 nucleotide residues in length. In particular embodiments,oligonucleotides may range from about 10 to about 100 nucleotides inlength. In various related embodiments, oligonucleotides, bothsingle-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 60 nucleotides, from about 15 to about 60nucleotides, from about 20 to about 50 nucleotides, from about 15 toabout 30 nucleotides, or from about 20 to about 30 nucleotides inlength.

In particular embodiments, an oligonucleotide (or a strand thereof) ofthe invention specifically hybridizes to or is complementary to a targetpolynucleotide sequence. The terms “specifically hybridizable” and“complementary” as used herein indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. In preferred embodiments,an oligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target sequence interferes with the normalfunction of the target sequence to cause a loss of utility or expressiontherefrom, and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or, in the case of in vitro assays, under conditions in whichthe assays are conducted. Thus, the oligonucleotide may include 1, 2, 3,or more base substitutions as compared to the region of a gene or mRNAsequence that it is targeting or to which it specifically hybridizes.

A. siRNA

The unmodified and modified siRNA molecules of the invention are capableof silencing SMAD4 gene expression. Each strand of the siRNA duplex istypically about 15 to about 60 nucleotides in length, preferably about15 to about 30 nucleotides in length. In certain embodiments, the siRNAcomprises at least one modified nucleotide. The modified siRNA isgenerally less immunostimulatory than a corresponding unmodified siRNAsequence and retains RNAi activity against the target gene of interest.In some embodiments, the modified siRNA contains at least one 2′OMepurine or pyrimidine nucleotide such as a 2′ OMe-guanosine, 2′OMe-uridine, 2′ OMe-adenosine, and/or 2′ OMe-cytosine nucleotide. Themodified nucleotides can be present in one strand (i.e., sense orantisense) or both strands of the siRNA. In some preferred embodiments,one or more of the uridine and/or guanosine nucleotides are modified(e.g., 2′OMe-modified) in one strand (i.e., sense or antisense) or bothstrands of the siRNA. In these embodiments, the modified siRNA canfurther comprise one or more modified (e.g., 2′OMe-modified) adenosineand/or modified (e.g., 2′OMe-modified) cytosine nucleotides. In otherpreferred embodiments, only uridine and/or guanosine nucleotides aremodified (e.g., 2′OMe-modified) in one strand (i.e., sense or antisense)or both strands of the siRNA. The siRNA sequences may have overhangs(e.g., 3′ or 5′ overhangs as described in Elbashir et al., Genes Dev.,15:188 (2001) or Nykänen et al., Cell, 107:309 (2001)), or may lackoverhangs (i.e., have blunt ends).

In particular embodiments, the selective incorporation of modifiednucleotides such as 2′OMe uridine and/or guanosine nucleotides into thedouble-stranded region of either or both strands of the siRNA reduces orcompletely abrogates the immune response to that siRNA molecule. Incertain instances, the immunostimulatory properties of specific siRNAsequences and their ability to silence gene expression can be balancedor optimized by the introduction of minimal and selective 2′OMemodifications within the double-stranded region of the siRNA duplex.This can be achieved at therapeutically viable siRNA doses withoutcytokine induction, toxicity, and off-target effects associated with theuse of unmodified siRNA.

The modified siRNA generally comprises from about 1% to about 100%(e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%) modified nucleotides in the double-stranded region ofthe siRNA duplex. In certain embodiments, one, two, three, four, five,six, seven, eight, nine, ten, or more of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides. Incertain other embodiments, some or all of the modified nucleotides inthe double-stranded region of the siRNA are 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more nucleotides apart from each other. In one preferredembodiment, none of the modified nucleotides in the double-strandedregion of the siRNA are adjacent to each other (e.g., there is a gap ofat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified nucleotides betweeneach modified nucleotide).

In some embodiments, less than about 50% (e.g., less than about 49%,48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, or 36%,preferably less than about 35%, 34%, 33%, 32%, 31%, or 30%) of thenucleotides in the double-stranded region of the siRNA comprise modified(e.g., 2′OMe) nucleotides. In one aspect of these embodiments, less thanabout 50% of the uridine and/or guanosine nucleotides in thedouble-stranded region of one or both strands of the siRNA areselectively (e.g., only) modified. In another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the siRNA,wherein the siRNA comprises at least one 2′OMe-guanosine nucleotide andat least one 2′OMe-uridine nucleotide, and wherein 2′OMe-guanosinenucleotides and 2′OMe-uridine nucleotides are the only 2′OMe nucleotidespresent in the double-stranded region. In yet another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the siRNA does not comprise 2′OMe-cytosine nucleotides in thedouble-stranded region. In a further aspect of these embodiments, lessthan about 50% of the nucleotides in the double-stranded region of thesiRNA comprise 2′OMe nucleotides, wherein the siRNA comprises 2′OMenucleotides in both strands of the siRNA, wherein the siRNA comprises atleast one 2′OMe-guanosine nucleotide and at least one 2′OMe-uridinenucleotide, and wherein the siRNA does not comprise 2′OMe-cytosinenucleotides in the double-stranded region. In another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the 2′OMe nucleotides in the double-stranded region are notadjacent to each other.

In other embodiments, from about 1% to about 50% (e.g., from about5%-50%, 10%-50%, 15%-50%, 20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%,45%-50%, 5%-45%, 10%-45%, 15%-45%, 20%-45%, 25%-45%, 30%-45%, 35%-45%,40%-45%, 5%-40%, 10%-40%, 15%-40%, 20%-40%, 25%-40%, 25%-39%, 25%-38%,25%-37%, 25%-36%, 26%-39%, 26%-38%, 26%-37%, 26%-36%, 27%-39%, 27%-38%,27%-37%, 27%-36%, 28%-39%, 28%-38%, 28%-37%, 28%-36%, 29%-39%, 29%-38%,29%-37%, 29%-36%, 30%-40%, 30%-39%, 30%-38%, 30%-37%, 30%-36%, 31%-39%,31%-38%, 31%-37%, 31%-36%, 32%-39%, 32%-38%, 32%-37%, 32%-36%, 33%-39%,33%-38%, 33%-37%, 33%-36%, 34%-39%, 34%-38%, 34%-37%, 34%-36%, 35%-40%,5%-35%, 10%-35%, 15%-35%, 20%-35%, 21%-35%, 22%-35%, 23%-35%, 24%-35%,25%-35%, 26%-35%, 27%-35%, 28%-35%, 29%-35%, 30%-35%, 31%-35%, 32%-35%,33%-35%, 34%-35%, 30%-34%, 31%-34%, 32%-34%, 33%-34%, 30%-33%, 31%-33%,32%-33%, 30%-32%, 31%-32%, 25%-34%, 25%-33%, 25%-32%, 25%-31%, 26%-34%,26%-33%, 26%-32%, 26%-31%, 27%-34%, 27%-33%, 27%-32%, 27%-31%, 28%-34%,28%-33%, 28%-32%, 28%-31%, 29%-34%, 29%-33%, 29%-32%, 29%-31%, 5%-30%,10%-30%, 15%-30%, 20%-34%, 20%-33%, 20%-32%, 20%-31%, 20%-30%, 21%-30%,22%-30%, 23%-30%, 24%-30%, 25%-30%, 25%-29%, 25%-28%, 25%-27%, 25%-26%,26%-30%, 26%-29%, 26%-28%, 26%-27%, 27%-30%, 27%-29%, 27%-28%, 28%-30%,28%-29%, 29%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-29%, 20%-28%, 20%-27%,20%-26%, 20%-25%, 5%-20%, 10%-20%, 15%-20%, 5%-15%, 10%-15%, or 5%-10%)of the nucleotides in the double-stranded region of the siRNA comprisemodified nucleotides. In one aspect of these embodiments, from about 1%to about 50% of the uridine and/or guanosine nucleotides in thedouble-stranded region of one or both strands of the siRNA areselectively (e.g., only) modified. In another aspect of theseembodiments, from about 1% to about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the siRNA,wherein the siRNA comprises at least one 2′OMe-guanosine nucleotide andat least one 2′OMe-uridine nucleotide, and wherein 2′OMe-guanosinenucleotides and 2′OMe-uridine nucleotides are the only 2′OMe nucleotidespresent in the double-stranded region. In yet another aspect of theseembodiments, from about 1% to about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the siRNA does not comprise 2′OMe-cytosine nucleotides in thedouble-stranded region. In a further aspect of these embodiments, fromabout 1% to about 50% of the nucleotides in the double-stranded regionof the siRNA comprise 2′OMe nucleotides, wherein the siRNA comprises2′OMe nucleotides in both strands of the siRNA, wherein the siRNAcomprises at least one 2′OMe-guanosine nucleotide and at least one2′OMe-uridine nucleotide, and wherein the siRNA does not comprise2′OMe-cytosine nucleotides in the double-stranded region. In anotheraspect of these embodiments, from about 1% to about 50% of thenucleotides in the double-stranded region of the siRNA comprise 2′OMenucleotides, wherein the siRNA comprises 2′OMe nucleotides in bothstrands of the modified siRNA, wherein the siRNA comprises 2′OMenucleotides selected from the group consisting of 2′OMe-guanosinenucleotides, 2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, andmixtures thereof, and wherein the 2′OMe nucleotides in thedouble-stranded region are not adjacent to each other.

Additional ranges, percentages, and patterns of modifications that maybe introduced into siRNA are described in U.S. Patent Publication No.20070135372, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

1. Selection of siRNA Sequences

Suitable siRNA sequences can be identified using any means known in theart. Typically, the methods described in Elbashir et al., Nature,411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) arecombined with rational design rules set forth in Reynolds et al., NatureBiotech., 22(3):326-330 (2004).

As a non-limiting example, the nucleotide sequence 3′ of the AUG startcodon of a transcript from the target gene of interest may be scannedfor dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, wherein N═C, G,or U) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). Thenucleotides immediately 3′ to the dinucleotide sequences are identifiedas potential siRNA sequences (i.e., a target sequence or a sense strandsequence). Typically, the 19, 21, 23, 25, 27, 29, 31, 33, 35, or morenucleotides immediately 3′ to the dinucleotide sequences are identifiedas potential siRNA sequences. In some embodiments, the dinucleotidesequence is an AA or NA sequence and the 19 nucleotides immediately 3′to the AA or NA dinucleotide are identified as potential siRNAsequences. siRNA sequences are usually spaced at different positionsalong the length of the target gene. To further enhance silencingefficiency of the siRNA sequences, potential siRNA sequences may beanalyzed to identify sites that do not contain regions of homology toother coding sequences, e.g., in the target cell or organism. Forexample, a suitable siRNA sequence of about 21 base pairs typically willnot have more than 16-17 contiguous base pairs of homology to codingsequences in the target cell or organism. If the siRNA sequences are tobe expressed from an RNA Pol III promoter, siRNA sequences lacking morethan 4 contiguous A's or T's are selected.

Once a potential siRNA sequence has been identified, a complementarysequence (i.e., an antisense strand sequence) can be designed. Apotential siRNA sequence can also be analyzed using a variety ofcriteria known in the art. For example, to enhance their silencingefficiency, the siRNA sequences may be analyzed by a rational designalgorithm to identify sequences that have one or more of the followingfeatures: (1) G/C content of about 25% to about 60% G/C; (2) at least 3A/Us at positions 15-19 of the sense strand; (3) no internal repeats;(4) an A at position 19 of the sense strand; (5) an A at position 3 ofthe sense strand; (6) a U at position 10 of the sense strand; (7) no G/Cat position 19 of the sense strand; and (8) no G at position 13 of thesense strand. siRNA design tools that incorporate algorithms that assignsuitable values of each of these features and are useful for selectionof siRNA can be found at, e.g.,http://ihome.ust.hk/˜bokcmho/siRNA/siRNA.html. One of skill in the artwill appreciate that sequences with one or more of the foregoingcharacteristics may be selected for further analysis and testing aspotential siRNA sequences.

Additionally, potential siRNA sequences with one or more of thefollowing criteria can often be eliminated as siRNA: (1) sequencescomprising a stretch of 4 or more of the same base in a row; (2)sequences comprising homopolymers of Gs (i.e., to reduce possiblenon-specific effects due to structural characteristics of thesepolymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC,AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in arow; and (5) sequences comprising direct repeats of 4 or more baseswithin the candidates resulting in internal fold-back structures.However, one of skill in the art will appreciate that sequences with oneor more of the foregoing characteristics may still be selected forfurther analysis and testing as potential siRNA sequences.

In some embodiments, potential siRNA sequences may be further analyzedbased on siRNA duplex asymmetry as described in, e.g., Khvorova et al.,Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208 (2003).In other embodiments, potential siRNA sequences may be further analyzedbased on secondary structure at the target site as described in, e.g.,Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example,secondary structure at the target site can be modeled using the Mfoldalgorithm (available at http://mfold.burnet.edu.au/rna_form) to selectsiRNA sequences which favor accessibility at the target site where lesssecondary structure in the form of base-pairing and stem-loops ispresent.

Once a potential siRNA sequence has been identified, the sequence can beanalyzed for the presence of any immunostimulatory properties, e.g.,using an in vitro cytokine assay or an in vivo animal model. Motifs inthe sense and/or antisense strand of the siRNA sequence such as GU-richmotifs (e.g., 5′-GU-3′,5′-UGU-3′,5′-GUGU-3′,5′-UGUGU-3′, etc.) can alsoprovide an indication of whether the sequence may be immunostimulatory.Once an siRNA molecule is found to be immunostimulatory, it can then bemodified to decrease its immunostimulatory properties as describedherein. As a non-limiting example, an siRNA sequence can be contactedwith a mammalian responder cell under conditions such that the cellproduces a detectable immune response to determine whether the siRNA isan immunostimulatory or a non-immunostimulatory siRNA. The mammalianresponder cell may be from a naïve mammal (i.e., a mammal that has notpreviously been in contact with the gene product of the siRNA sequence).The mammalian responder cell may be, e.g., a peripheral bloodmononuclear cell (PBMC), a macrophage, and the like. The detectableimmune response may comprise production of a cytokine or growth factorsuch as, e.g., TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-12, or a combinationthereof. An siRNA molecule identified as being immunostimulatory canthen be modified to decrease its immunostimulatory properties byreplacing at least one of the nucleotides on the sense and/or antisensestrand with modified nucleotides. For example, less than about 30%(e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) of thenucleotides in the double-stranded region of the siRNA duplex can bereplaced with modified nucleotides such as 2′OMe nucleotides. Themodified siRNA can then be contacted with a mammalian responder cell asdescribed above to confirm that its immunostimulatory properties havebeen reduced or abrogated.

Suitable in vitro assays for detecting an immune response include, butare not limited to, the double monoclonal antibody sandwich immunoassaytechnique of David et al. (U.S. Pat. No. 4,376,110);monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkhamand Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone,Edinburgh (1970)); the “Western blot” method of Gordon et al. (U.S. Pat.No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assays(ELISA) as described, for example, by Raines et al., J. Biol. Chem.,257:5154-5160 (1982); immunocytochemical techniques, including the useof fluorochromes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980));and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad.Sci. USA, 81:2396-2400 (1984)). In addition to the immunoassaysdescribed above, a number of other immunoassays are available, includingthose described in U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Thedisclosures of these references are herein incorporated by reference intheir entirety for all purposes.

A non-limiting example of an in vivo model for detecting an immuneresponse includes an in vivo mouse cytokine induction assay as describedin, e.g., Judge et al., Mol. Ther., 13:494-505 (2006). In certainembodiments, the assay that can be performed as follows: (1) siRNA canbe administered by standard intravenous injection in the lateral tailvein; (2) blood can be collected by cardiac puncture about 6 hours afteradministration and processed as plasma for cytokine analysis; and (3)cytokines can be quantified using sandwich ELISA kits according to themanufacturer's instructions (e.g., mouse and human IFN-α (PBLBiomedical; Piscataway, N.J.); human IL-6 and TNF-α (eBioscience; SanDiego, Calif.); and mouse IL-6, TNF-α, and IFN-γ(BD Biosciences; SanDiego, Calif.)).

Monoclonal antibodies that specifically bind cytokines and growthfactors are commercially available from multiple sources and can begenerated using methods known in the art (see, e.g., Kohler et al.,Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)).Generation of monoclonal antibodies has been previously described andcan be accomplished by any means known in the art (Buhring et al., inHybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, themonoclonal antibody is labeled (e.g., with any composition detectable byspectroscopic, photochemical, biochemical, electrical, optical, orchemical means) to facilitate detection.

2. Generating siRNA Molecules

siRNA can be provided in several forms including, e.g., as one or moreisolated small-interfering RNA (siRNA) duplexes, as longerdouble-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from atranscriptional cassette in a DNA plasmid. In some embodiments, siRNAmay be produced enzymatically or by partial/total organic synthesis, andmodified ribonucleotides can be introduced by in vitro enzymatic ororganic synthesis. In certain instances, each strand is preparedchemically. Methods of synthesizing RNA molecules are known in the art,e.g., the chemical synthesis methods as described in Verma and Eckstein(1998) or as described herein.

An RNA population can be used to provide long precursor RNAs, or longprecursor RNAs that have substantial or complete identity to a selectedtarget sequence can be used to make the siRNA. The RNAs can be isolatedfrom cells or tissue, synthesized, and/or cloned according to methodswell known to those of skill in the art. The RNA can be a mixedpopulation (obtained from cells or tissue, transcribed from cDNA,subtracted, selected, etc.), or can represent a single target sequence.RNA can be naturally occurring (e.g., isolated from tissue or cellsamples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCRproducts or a cloned cDNA), or chemically synthesized.

To form a long dsRNA, for synthetic RNAs, the complement is alsotranscribed in vitro and hybridized to form a dsRNA. If a naturallyoccurring RNA population is used, the RNA complements are also provided(e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g.,by transcribing cDNAs corresponding to the RNA population, or by usingRNA polymerases. The precursor RNAs are then hybridized to form doublestranded RNAs for digestion. The dsRNAs can be directly administered toa subject or can be digested in vitro prior to administration.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see,U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Expressionlibraries are also well known to those of skill in the art. Additionalbasic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994). The disclosures of these references are herein incorporatedby reference in their entirety for all purposes.

Preferably, siRNA are chemically synthesized. The oligonucleotides thatcomprise the siRNA molecules of the invention can be synthesized usingany of a variety of techniques known in the art, such as those describedin Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al.,Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res.,23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59(1997). The synthesis of oligonucleotides makes use of common nucleicacid protecting and coupling groups, such as dimethoxytrityl at the5′-end and phosphoramidites at the 3′-end. As a non-limiting example,small scale syntheses can be conducted on an Applied Biosystemssynthesizer using a 0.2 mol scale protocol. Alternatively, syntheses atthe 0.2 mol scale can be performed on a 96-well plate synthesizer fromProtogene (Palo Alto, Calif.). However, a larger or smaller scale ofsynthesis is also within the scope of this invention. Suitable reagentsfor oligonucleotide synthesis, methods for RNA deprotection, and methodsfor RNA purification are known to those of skill in the art.

siRNA molecules can also be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousoligonucleotide fragment or strand separated by a cleavable linker thatis subsequently cleaved to provide separate fragments or strands thathybridize to form the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker. The tandem synthesis of siRNA can bereadily adapted to both multiwell/multiplate synthesis plaSMAD4orms aswell as large scale synthesis plaSMAD4orms employing batch reactors,synthesis columns, and the like. Alternatively, siRNA molecules can beassembled from two distinct oligonucleotides, wherein oneoligonucleotide comprises the sense strand and the other comprises theantisense strand of the siRNA. For example, each strand can besynthesized separately and joined together by hybridization or ligationfollowing synthesis and/or deprotection. In certain other instances,siRNA molecules can be synthesized as a single continuousoligonucleotide fragment, where the self-complementary sense andantisense regions hybridize to form an siRNA duplex having hairpinsecondary structure.

3. Modifying siRNA Sequences

In certain aspects, siRNA molecules comprise a duplex having two strandsand at least one modified nucleotide in the double-stranded region,wherein each strand is about 15 to about 60 nucleotides in length.Advantageously, the modified siRNA is less immunostimulatory than acorresponding unmodified siRNA sequence, but retains the capability ofsilencing the expression of a target sequence. In preferred embodiments,the degree of chemical modifications introduced into the siRNA moleculestrikes a balance between reduction or abrogation of theimmunostimulatory properties of the siRNA and retention of RNAiactivity. As a non-limiting example, an siRNA molecule that targets agene of interest can be minimally modified (e.g., less than about 30%,25%, 20%, 15%, 10%, or 5% modified) at selective uridine and/orguanosine nucleotides within the siRNA duplex to eliminate the immuneresponse generated by the siRNA while retaining its capability tosilence target gene expression.

Examples of modified nucleotides suitable for use in the inventioninclude, but are not limited to, ribonucleotides having a 2′-O-methyl(2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl,2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.Modified nucleotides having a Northern conformation such as thosedescribed in, e.g., Saenger, Principles of Nucleic Acid Structure,Springer-Verlag Ed. (1984), are also suitable for use in siRNAmolecules. Such modified nucleotides include, without limitation, lockednucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl)(MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro (2′Cl) nucleotides, and 2′-azidonucleotides. In certain instances, the siRNA molecules described hereininclude one or more G-clamp nucleotides. A G-clamp nucleotide refers toa modified cytosine analog wherein the modifications confer the abilityto hydrogen bond both Watson-Crick and Hoogsteen faces of acomplementary guanine nucleotide within a duplex (see, e.g., Lin et al.,J. Am. Chem. Soc., 120:8531-8532 (1998)). In addition, nucleotideshaving a nucleotide base analog such as, for example, C-phenyl,C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, andnitroazole derivatives such as 3-nitropyrrole, 4-nitroindole,5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res.,29:2437-2447 (2001)) can be incorporated into siRNA molecules.

In certain embodiments, siRNA molecules may further comprise one or morechemical modifications such as terminal cap moieties, phosphate backbonemodifications, and the like. Examples of terminal cap moieties include,without limitation, inverted deoxy abasic residues, glycerylmodifications, 4′,5′-methylene nucleotides, 1-(β-D-erythrofuranosyl)nucleotides, 4′-thio nucleotides, carbocyclic nucleotides,1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modifiedbase nucleotides, threo-pentofuranosyl nucleotides, acyclic 3′,4′-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties,3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties,3′-2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties,5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties,5′-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropylphosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate,hydroxypropyl phosphate, 1,4-butanediol phosphate, 3′-phosphoramidate,5′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate,and bridging or non-bridging methylphosphonate or 5′-mercapto moieties(see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron49:1925 (1993)). Non-limiting examples of phosphate backbonemodifications (i.e., resulting in modified internucleotide linkages)include phosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate, carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker etal., Nucleic Acid Analogues: Synthesis and Properties, in ModernSynthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39 (1994)). Such chemical modifications canoccur at the 5′-end and/or 3′-end of the sense strand, antisense strand,or both strands of the siRNA. The disclosures of these references areherein incorporated by reference in their entirety for all purposes.

In some embodiments, the sense and/or antisense strand of the siRNAmolecule can further comprise a 3′-terminal overhang having about 1 toabout 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides, modified (e.g.,2′OMe) and/or unmodified uridine ribonucleotides, and/or any othercombination of modified (e.g., 2′OMe) and unmodified nucleotides.

Additional examples of modified nucleotides and types of chemicalmodifications that can be introduced into siRNA molecules are described,e.g., in UK Patent No. GB 2,397,818 B and U.S. Patent Publication Nos.20040192626, 20050282188, and 20070135372, the disclosures of which areherein incorporated by reference in their entirety for all purposes.

The siRNA molecules described herein can optionally comprise one or morenon-nucleotides in one or both strands of the siRNA. As used herein, theterm “non-nucleotide” refers to any group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including sugar and/or phosphate substitutions, andallows the remaining bases to exhibit their activity. The group orcompound is abasic in that it does not contain a commonly recognizednucleotide base such as adenosine, guanine, cytosine, uracil, or thymineand therefore lacks a base at the 1′-position.

In other embodiments, chemical modification of the siRNA comprisesattaching a conjugate to the siRNA molecule. The conjugate can beattached at the 5′ and/or 3′-end of the sense and/or antisense strand ofthe siRNA via a covalent attachment such as, e.g., a biodegradablelinker. The conjugate can also be attached to the siRNA, e.g., through acarbamate group or other linking group (see, e.g., U.S. PatentPublication Nos. 20050074771, 20050043219, and 20050158727). In certaininstances, the conjugate is a molecule that facilitates the delivery ofthe siRNA into a cell. Examples of conjugate molecules suitable forattachment to siRNA include, without limitation, steroids such ascholesterol, glycols such as polyethylene glycol (PEG), human serumalbumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates(e.g., folic acid, folate analogs and derivatives thereof), sugars(e.g., galactose, galactosamine, N-acetyl galactosamine, glucose,mannose, fructose, fucose, etc.), phospholipids, peptides, ligands forcellular receptors capable of mediating cellular uptake, andcombinations thereof (see, e.g., U.S. Patent Publication Nos.20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423).Other examples include the lipophilic moiety, vitamin, polymer, peptide,protein, nucleic acid, small molecule, oligosaccharide, carbohydratecluster, intercalator, minor groove binder, cleaving agent, andcross-linking agent conjugate molecules described in U.S. PatentPublication Nos. 20050119470 and 20050107325. Yet other examples includethe 2′-O-alkyl amine, 2′-alkoxyalkyl amine, polyamine, C5-cationicmodified pyrimidine, cationic peptide, guanidinium group, amidininiumgroup, cationic amino acid conjugate molecules described in U.S. PatentPublication No. 20050153337. Additional examples include the hydrophobicgroup, membrane active compound, cell penetrating compound, celltargeting signal, interaction modifier, and steric stabilizer conjugatemolecules described in U.S. Patent Publication No. 20040167090. Furtherexamples include the conjugate molecules described in U.S. PatentPublication No. 20050239739. The type of conjugate used and the extentof conjugation to the siRNA molecule can be evaluated for improvedpharmacokinetic profiles, bioavailability, and/or stability of the siRNAwhile retaining RNAi activity. As such, one skilled in the art canscreen siRNA molecules having various conjugates attached thereto toidentify ones having improved properties and full RNAi activity usingany of a variety of well-known in vitro cell culture or in vivo animalmodels. The disclosures of the above-described patent documents areherein incorporated by reference in their entirety for all purposes.

4. Exemplary siRNA Embodiments

In some embodiments, each strand of the siRNA molecule comprises fromabout 15 to about 60 nucleotides in length (e.g., about 15-60, 15-50,15-40, 15-30, 15-25, or 19-25 nucleotides in length, or 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In one particularembodiment, the siRNA is chemically synthesized. The siRNA molecules ofthe invention are capable of silencing the expression of a targetsequence in vitro and/or in vivo.

In other embodiments, the siRNA comprises at least one modifiednucleotide. In certain embodiments, the siRNA comprises one, two, three,four, five, six, seven, eight, nine, ten, or more modified nucleotidesin the double-stranded region. In particular embodiments, less thanabout 50% (e.g., less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, or 5%) of the nucleotides in the double-stranded region of thesiRNA comprise modified nucleotides. In preferred embodiments, fromabout 1% to about 50% (e.g., from about 5%-50%, 10%-50%, 15%-50%,20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%, 45%-50%, 5%-45%, 10%-45%,15%-45%, 20%-45%, 25%-45%, 30%-45%, 35%-45%, 40%-45%, 5%-40%, 10%-40%,15%-40%, 20%-40%, 25%-40%, 30%-40%, 35%-40%, 5%-35%, 10%-35%, 15%-35%,20%-35%, 25%-35%, 30%-35%, 5%-30%, 10%-30%, 15%-30%, 20%-30%, 25%-30%,5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, 15%-20%, 5%-15%,10%-15%, or 5%-10%) of the nucleotides in the double-stranded region ofthe siRNA comprise modified nucleotides.

In further embodiments, the siRNA comprises modified nucleotidesincluding, but not limited to, 2′-O-methyl (2′OMe) nucleotides,2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, and mixtures thereof. In preferred embodiments, the siRNAcomprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, e.g., 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosine nucleotides, ormixtures thereof. In one particular embodiment, the siRNA comprises atleast one 2′OMe-guanosine nucleotide, 2′OMe-uridine nucleotide, ormixtures thereof. In certain instances, the siRNA does not comprise2′OMe-cytosine nucleotides. In other embodiments, the siRNA comprises ahairpin loop structure.

In certain embodiments, the siRNA comprises modified nucleotides in onestrand (i.e., sense or antisense) or both strands of the double-strandedregion of the siRNA molecule. Preferably, uridine and/or guanosinenucleotides are modified at selective positions in the double-strandedregion of the siRNA duplex. With regard to uridine nucleotidemodifications, at least one, two, three, four, five, six, or more of theuridine nucleotides in the sense and/or antisense strand can be amodified uridine nucleotide such as a 2′OMe-uridine nucleotide. In someembodiments, every uridine nucleotide in the sense and/or antisensestrand is a 2′OMe-uridine nucleotide. With regard to guanosinenucleotide modifications, at least one, two, three, four, five, six, ormore of the guanosine nucleotides in the sense and/or antisense strandcan be a modified guanosine nucleotide such as a 2′OMe-guanosinenucleotide. In some embodiments, every guanosine nucleotide in the senseand/or antisense strand is a 2′OMe-guanosine nucleotide.

In certain embodiments, at least one, two, three, four, five, six,seven, or more 5′-GU-3′ motifs in an siRNA sequence may be modified,e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/orby introducing modified nucleotides such as 2′OMe nucleotides. The5′-GU-3′ motif can be in the sense strand, the antisense strand, or bothstrands of the siRNA sequence. The 5′-GU-3′ motifs may be adjacent toeach other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more nucleotides.

In some embodiments, a modified siRNA molecule is less immunostimulatorythan a corresponding unmodified siRNA sequence. In such embodiments, themodified siRNA molecule with reduced immunostimulatory propertiesadvantageously retains RNAi activity against the target sequence. Inanother embodiment, the immunostimulatory properties of the modifiedsiRNA molecule and its ability to silence target gene expression can bebalanced or optimized by the introduction of minimal and selective 2′OMemodifications within the siRNA sequence such as, e.g., within thedouble-stranded region of the siRNA duplex. In certain instances, themodified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that the immunostimulatory properties of the modifiedsiRNA molecule and the corresponding unmodified siRNA molecule can bedetermined by, for example, measuring INF-α and/or IL-6 levels fromabout two to about twelve hours after systemic administration in amammal or transfection of a mammalian responder cell using anappropriate lipid-based delivery system (such as the SNALP deliverysystem disclosed herein).

In other embodiments, a modified siRNA molecule has an IC50 (i.e.,half-maximal inhibitory concentration) less than or equal to ten-foldthat of the corresponding unmodified siRNA (i.e., the modified siRNA hasan IC50 that is less than or equal to ten-times the IC50 of thecorresponding unmodified siRNA). In other embodiments, the modifiedsiRNA has an IC50 less than or equal to three-fold that of thecorresponding unmodified siRNA sequence. In yet other embodiments, themodified siRNA has an IC50 less than or equal to two-fold that of thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that a dose-response curve can be generated and theIC50 values for the modified siRNA and the corresponding unmodifiedsiRNA can be readily determined using methods known to those of skill inthe art.

In another embodiment, an unmodified or modified siRNA molecule iscapable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% of the expression of the target sequencerelative to a negative control (e.g., buffer only, an siRNA sequencethat targets a different gene, a scrambled siRNA sequence, etc.).

In yet another embodiment, a modified siRNA molecule is capable ofsilencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% of the expression of the target sequence relative tothe corresponding unmodified siRNA sequence.

In some embodiments, the siRNA molecule does not comprise phosphatebackbone modifications, e.g., in the sense and/or antisense strand ofthe double-stranded region. In other embodiments, the siRNA comprisesone, two, three, four, or more phosphate backbone modifications, e.g.,in the sense and/or antisense strand of the double-stranded region. Inpreferred embodiments, the siRNA does not comprise phosphate backbonemodifications.

In further embodiments, the siRNA does not comprise 2′-deoxynucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region. In yet further embodiments, the siRNA comprisesone, two, three, four, or more 2′-deoxy nucleotides, e.g., in the senseand/or antisense strand of the double-stranded region. In preferredembodiments, the siRNA does not comprise 2′-deoxy nucleotides.

In certain instances, the nucleotide at the 3′-end of thedouble-stranded region in the sense and/or antisense strand is not amodified nucleotide. In certain other instances, the nucleotides nearthe 3′-end (e.g., within one, two, three, or four nucleotides of the3′-end) of the double-stranded region in the sense and/or antisensestrand are not modified nucleotides.

The siRNA molecules described herein may have 3′ overhangs of one, two,three, four, or more nucleotides on one or both sides of thedouble-stranded region, or may lack overhangs (i.e., have blunt ends) onone or both sides of the double-stranded region. In certain embodiments,the 3′ overhang on the sense and/or antisense strand independentlycomprises one, two, three, four, or more modified nucleotides such as2′OMe nucleotides and/or any other modified nucleotide described hereinor known in the art.

In particular embodiments, siRNAs targeting SMAD4 RNA or SMAD4 mRNA areadministered using a carrier system such as a nucleic acid-lipidparticle. In a preferred embodiment, the nucleic acid-lipid particlecomprises: (a) one or more siRNA molecules targeting SMAD4; (b) acationic lipid (e.g., DLinDMA, DLenDMA, DLin-K-C2-DMA, and/orγ-DLenDMA); and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/orcholesterol). In certain instances, the nucleic acid-lipid particle mayfurther comprise a conjugated lipid that prevents aggregation ofparticles (e.g., PEG-DAA and/or POZ-DAA).

In addition to its utility in silencing the expression of any of theabove-described SMAD4 genes for therapeutic purposes, the siRNAdescribed herein are also useful in research and developmentapplications as well as diagnostic, prophylactic, prognostic, clinical,and other healthcare applications. As a non-limiting example, the siRNAcan be used in target validation studies directed at testing whether aspecific member of the SMAD4 gene family has the potential to be atherapeutic target.

B. Dicer-Substrate dsRNA

As used herein, the term “Dicer-substrate dsRNA” or “precursor RNAimolecule” is intended to include any precursor molecule that isprocessed in vivo by Dicer to produce an active siRNA which isincorporated into the RISC complex for RNA interference of a targetgene.

In one embodiment, the Dicer-substrate dsRNA has a length sufficientsuch that it is processed by Dicer to produce an siRNA. According tothis embodiment, the Dicer-substrate dsRNA comprises (i) a firstoligonucleotide sequence (also termed the sense strand) that is betweenabout 25 and about 60 nucleotides in length (e.g., about 25-60, 25-55,25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), preferablybetween about 25 and about 30 nucleotides in length (e.g., 25, 26, 27,28, 29, or 30 nucleotides in length), and (ii) a second oligonucleotidesequence (also termed the antisense strand) that anneals to the firstsequence under biological conditions, such as the conditions found inthe cytoplasm of a cell. The second oligonucleotide sequence may bebetween about 25 and about 60 nucleotides in length (e.g., about 25-60,25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), andis preferably between about 25 and about 30 nucleotides in length (e.g.,25, 26, 27, 28, 29, or 30 nucleotides in length). In addition, a regionof one of the sequences, particularly of the antisense strand, of theDicer-substrate dsRNA has a sequence length of at least about 19nucleotides, for example, from about 19 to about 60 nucleotides (e.g.,about 19-60, 19-55, 19-50, 19-45, 19-40, 19-35, 19-30, or 19-25nucleotides), preferably from about 19 to about 23 nucleotides (e.g.,19, 20, 21, 22, or 23 nucleotides) that are sufficiently complementaryto a nucleotide sequence of the RNA produced from the target gene totrigger an RNAi response.

In a second embodiment, the Dicer-substrate dsRNA has several propertieswhich enhance its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and has at least one of the following properties: (i)the dsRNA is asymmetric, e.g., has a 3′-overhang on the antisensestrand; and/or (ii) the dsRNA has a modified 3′-end on the sense strandto direct orientation of Dicer binding and processing of the dsRNA to anactive siRNA. According to this latter embodiment, the sense strandcomprises from about 22 to about 28 nucleotides and the antisense strandcomprises from about 24 to about 30 nucleotides.

In one embodiment, the Dicer-substrate dsRNA has an overhang on the3′-end of the antisense strand. In another embodiment, the sense strandis modified for Dicer binding and processing by suitable modifierslocated at the 3′-end of the sense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides, and thelike, and sterically hindered molecules such as fluorescent moleculesand the like. When nucleotide modifiers are used, they replaceribonucleotides in the dsRNA such that the length of the dsRNA does notchange. In another embodiment, the Dicer-substrate dsRNA has an overhangon the 3′-end of the antisense strand and the sense strand is modifiedfor Dicer processing. In another embodiment, the 5′-end of the sensestrand has a phosphate. In another embodiment, the 5′-end of theantisense strand has a phosphate. In another embodiment, the antisensestrand or the sense strand or both strands have one or more 2′-O -methyl(2′OMe) modified nucleotides. In another embodiment, the antisensestrand contains 2′OMe modified nucleotides. In another embodiment, theantisense stand contains a 3′-overhang that is comprised of 2′OMemodified nucleotides. The antisense strand could also include additional2′OMe modified nucleotides. The sense and antisense strands anneal underbiological conditions, such as the conditions found in the cytoplasm ofa cell. In addition, a region of one of the sequences, particularly ofthe antisense strand, of the Dicer-substrate dsRNA has a sequence lengthof at least about 19 nucleotides, wherein these nucleotides are in the21-nucleotide region adjacent to the 3′-end of the antisense strand andare sufficiently complementary to a nucleotide sequence of the RNAproduced from the target gene. Further, in accordance with thisembodiment, the Dicer-substrate dsRNA may also have one or more of thefollowing additional properties: (a) the antisense strand has a rightshift from the typical 21-mer (i.e., the antisense strand includesnucleotides on the right side of the molecule when compared to thetypical 21-mer); (b) the strands may not be completely complementary,i.e., the strands may contain simple mismatch pairings; and (c) basemodifications such as locked nucleic acid(s) may be included in the5′-end of the sense strand.

In a third embodiment, the sense strand comprises from about 25 to about28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides), wherein the 2nucleotides on the 3′-end of the sense strand are deoxyribonucleotides.The sense strand contains a phosphate at the 5′-end. The antisensestrand comprises from about 26 to about 30 nucleotides (e.g., 26, 27,28, 29, or 30 nucleotides) and contains a 3′-overhang of 1-4nucleotides. The nucleotides comprising the 3′-overhang are modifiedwith 2′OMe modified ribonucleotides. The antisense strand containsalternating 2′OMe modified nucleotides beginning at the first monomer ofthe antisense strand adjacent to the 3′-overhang, and extending 15-19nucleotides from the first monomer adjacent to the 3′-overhang. Forexample, for a 27-nucleotide antisense strand and counting the firstbase at the 5′-end of the antisense strand as position number 1, 2′OMemodifications would be placed at bases 9, 11, 13, 15, 17, 19, 21, 23,25, 26, and 27. In one embodiment, the Dicer-substrate dsRNA has thefollowing structure:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ (SEQ ID NO: 1)3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′ (SEQ ID NO: 2)

wherein “X”=RNA, “p”=a phosphate group, “X”=2′OMe RNA, “Y” is anoverhang domain comprised of 1, 2, 3, or 4 RNA monomers that areoptionally 2′OMe RNA monomers, and “D”=DNA. The top strand is the sensestrand, and the bottom strand is the antisense strand.

In a fourth embodiment, the Dicer-substrate dsRNA has several propertieswhich enhance its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and at least one of the following properties: (i) thedsRNA is asymmetric, e.g., has a 3′-overhang on the sense strand; and(ii) the dsRNA has a modified 3′-end on the antisense strand to directorientation of Dicer binding and processing of the dsRNA to an activesiRNA. According to this embodiment, the sense strand comprises fromabout 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30nucleotides) and the antisense strand comprises from about 22 to about28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides). In oneembodiment, the Dicer-substrate dsRNA has an overhang on the 3′-end ofthe sense strand. In another embodiment, the antisense strand ismodified for Dicer binding and processing by suitable modifiers locatedat the 3′-end of the antisense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides, and thelike, and sterically hindered molecules such as fluorescent moleculesand the like. When nucleotide modifiers are used, they replaceribonucleotides in the dsRNA such that the length of the dsRNA does notchange. In another embodiment, the dsRNA has an overhang on the 3′-endof the sense strand and the antisense strand is modified for Dicerprocessing. In one embodiment, the antisense strand has a 5′-phosphate.The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are adjacent to the 3′-end of antisense strand and aresufficiently complementary to a nucleotide sequence of the RNA producedfrom the target gene. Further, in accordance with this embodiment, theDicer-substrate dsRNA may also have one or more of the followingadditional properties: (a) the antisense strand has a left shift fromthe typical 21-mer (i.e., the antisense strand includes nucleotides onthe left side of the molecule when compared to the typical 21-mer); and(b) the strands may not be completely complementary, i.e., the strandsmay contain simple mismatch pairings.

In a preferred embodiment, the Dicer-substrate dsRNA has an asymmetricstructure, with the sense strand having a 25-base pair length, and theantisense strand having a 27-base pair length with a 2 base 3′-overhang.In certain instances, this dsRNA having an asymmetric structure furthercontains 2 deoxynucleotides at the 3′-end of the sense strand in placeof two of the ribonucleotides. In certain other instances, this dsRNAhaving an asymmetric structure further contains 2′OMe modifications atpositions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense strand(wherein the first base at the 5′-end of the antisense strand isposition 1). In certain additional instances, this dsRNA having anasymmetric structure further contains a 3′-overhang on the antisensestrand comprising 1, 2, 3, or 4 2′OMe nucleotides (e.g., a 3′-overhangof 2′OMe nucleotides at positions 26 and 27 on the antisense strand).

In another embodiment, Dicer-substrate dsRNAs may be designed by firstselecting an antisense strand siRNA sequence having a length of at least19 nucleotides. In some instances, the antisense siRNA is modified toinclude about 5 to about 11 ribonucleotides on the 5′-end to provide alength of about 24 to about 30 nucleotides. When the antisense strandhas a length of 21 nucleotides, 3-9, preferably 4-7, or more preferably6 nucleotides may be added on the 5′-end. Although the addedribonucleotides may be complementary to the target gene sequence, fullcomplementarity between the target sequence and the antisense siRNA isnot required. That is, the resultant antisense siRNA is sufficientlycomplementary with the target sequence. A sense strand is then producedthat has about 22 to about 28 nucleotides. The sense strand issubstantially complementary with the antisense strand to anneal to theantisense strand under biological conditions. In one embodiment, thesense strand is synthesized to contain a modified 3′-end to direct Dicerprocessing of the antisense strand. In another embodiment, the antisensestrand of the dsRNA has a 3′-overhang. In a further embodiment, thesense strand is synthesized to contain a modified 3′-end for Dicerbinding and processing and the antisense strand of the dsRNA has a3′-overhang.

In a related embodiment, the antisense siRNA may be modified to includeabout 1 to about 9 ribonucleotides on the 5′-end to provide a length ofabout 22 to about 28 nucleotides. When the antisense strand has a lengthof 21 nucleotides, 1-7, preferably 2-5, or more preferably 4ribonucleotides may be added on the 3′-end. The added ribonucleotidesmay have any sequence. Although the added ribonucleotides may becomplementary to the target gene sequence, full complementarity betweenthe target sequence and the antisense siRNA is not required. That is,the resultant antisense siRNA is sufficiently complementary with thetarget sequence. A sense strand is then produced that has about 24 toabout 30 nucleotides. The sense strand is substantially complementarywith the antisense strand to anneal to the antisense strand underbiological conditions. In one embodiment, the antisense strand issynthesized to contain a modified 3′-end to direct Dicer processing. Inanother embodiment, the sense strand of the dsRNA has a 3′-overhang. Ina further embodiment, the antisense strand is synthesized to contain amodified 3′-end for Dicer binding and processing and the sense strand ofthe dsRNA has a 3′-overhang.

Suitable Dicer-substrate dsRNA sequences can be identified, synthesized,and modified using any means known in the art for designing,synthesizing, and modifying siRNA sequences. In certain embodiments,Dicer-substrate dsRNAs of the invention may silence SMAD4 geneexpression. In particular embodiments, Dicer-substrate dsRNAs targetingSMAD4 mRNA are administered using a carrier system such as a nucleicacid-lipid particle. In a preferred embodiment, the nucleic acid-lipidparticle comprises: (a) one or more Dicer-substrate dsRNA moleculestargeting SMAD4 gene expression; (b) a cationic lipid (e.g., DLinDMA,DLenDMA, DLin-K-C2-DMA, and/or γ-DLenDMA); and (c) a non-cationic lipid(e.g., DPPC, DSPC, DSPE, and/or cholesterol). In certain instances, thenucleic acid-lipid particle may further comprise a conjugated lipid thatprevents aggregation of particles (e.g., PEG-DAA and/or POZ-DAA).

Additional embodiments related to the Dicer-substrate dsRNAs of theinvention, as well as methods of designing and synthesizing such dsRNAs,are described in U.S. Patent Publication Nos. 20050244858, 20050277610,and 20070265220, 2011/0071208, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

C. shRNA

A “small hairpin RNA” or “short hairpin RNA” or “shRNA” includes a shortRNA sequence that makes a tight hairpin turn that can be used to silencegene expression via RNA interference. The shRNAs of the invention may bechemically synthesized or transcribed from a transcriptional cassette ina DNA plasmid. The shRNA hairpin structure is cleaved by the cellularmachinery into siRNA, which is then bound to the RNA-induced silencingcomplex (RISC).

The shRNAs of the invention are typically about 15-60, 15-50, or 15-40(duplex) nucleotides in length, more typically about 15-30, 15-25, or19-25 (duplex) nucleotides in length, and are preferably about 20-24,21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementarysequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30,15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or21-23 nucleotides in length, and the double-stranded shRNA is about15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length,preferably about 18-22, 19-20, or 19-21 base pairs in length). shRNAduplexes may comprise 3′ overhangs of about 1 to about 4 nucleotides orabout 2 to about 3 nucleotides on the antisense strand and/or5′-phosphate termini on the sense strand. In some embodiments, the shRNAcomprises a sense strand and/or antisense strand sequence of from about15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50,15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), preferablyfrom about 19 to about 40 nucleotides in length (e.g., about 19-40,19-35, 19-30, or 19-25 nucleotides in length), more preferably fromabout 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23nucleotides in length).

Non-limiting examples of shRNA include a double-stranded polynucleotidemolecule assembled from a single-stranded molecule, where the sense andantisense regions are linked by a nucleic acid-based or non-nucleicacid-based linker; and a double-stranded polynucleotide molecule with ahairpin secondary structure having self-complementary sense andantisense regions. In preferred embodiments, the sense and antisensestrands of the shRNA are linked by a loop structure comprising fromabout 1 to about 25 nucleotides, from about 2 to about 20 nucleotides,from about 4 to about 15 nucleotides, from about 5 to about 12nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.

Suitable shRNA sequences can be identified, synthesized, and modifiedusing any means known in the art for designing, synthesizing, andmodifying siRNA sequences. In certain embodiments, shRNAs of theinvention may silence SMAD4 gene expression. In particular embodiments,shRNAs targeting SMAD4 mRNA are administered using a carrier system suchas a nucleic acid-lipid particle. In a preferred embodiment, the nucleicacid-lipid particle comprises: (a) one or more shRNA molecules targetingSMAD4 gene expression; (b) a cationic lipid (e.g., DLinDMA, DLenDMA,DLin-K-C2-DMA, and/or γ-DLenDMA); and (c) a non-cationic lipid (e.g.,DPPC, DSPC, DSPE, and/or cholesterol). In certain instances, the nucleicacid-lipid particle may further comprise a conjugated lipid thatprevents aggregation of particles (e.g., PEG-DAA and/or POZ-DAA).

Additional embodiments related to the shRNAs of the invention, as wellas methods of designing and synthesizing such shRNAs, are described inU.S. patent application publication number 2011/0071208, the disclosureof which is herein incorporated by reference in its entirety for allpurposes.

D. aiRNA

Like siRNA, asymmetrical interfering RNA (aiRNA) can recruit theRNA-induced silencing complex (RISC) and lead to effective silencing ofa variety of genes in mammalian cells by mediating sequence-specificcleavage of the target sequence between nucleotide 10 and 11 relative tothe 5′ end of the antisense strand (Sun et al., Nat. Biotech.,26:1379-1382 (2008)). Typically, an aiRNA molecule comprises a short RNAduplex having a sense strand and an antisense strand, wherein the duplexcontains overhangs at the 3′ and 5′ ends of the antisense strand. TheaiRNA is generally asymmetric because the sense strand is shorter onboth ends when compared to the complementary antisense strand. In someaspects, aiRNA molecules may be designed, synthesized, and annealedunder conditions similar to those used for siRNA molecules. As anon-limiting example, aiRNA sequences may be selected and generatedusing the methods described above for selecting siRNA sequences.

In another embodiment, aiRNA duplexes of various lengths (e.g., about10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more typically12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs) may be designed withoverhangs at the 3′ and 5′ ends of the antisense strand to target anmRNA of interest. In certain instances, the sense strand of the aiRNAmolecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or20 nucleotides in length. In certain other instances, the antisensestrand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and is preferably about 20-24, 21-22, or 21-23 nucleotides inlength.

In some embodiments, the 5′ antisense overhang contains one, two, three,four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”, etc.).In other embodiments, the 3′ antisense overhang contains one, two,three, four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”,etc.). In certain aspects, the aiRNA molecules described herein maycomprise one or more modified nucleotides, e.g., in the double-stranded(duplex) region and/or in the antisense overhangs. As a non-limitingexample, aiRNA sequences may comprise one or more of the modifiednucleotides described above for siRNA sequences. In a preferredembodiment, the aiRNA molecule comprises 2′OMe nucleotides such as, forexample, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, ormixtures thereof.

In certain embodiments, aiRNA molecules may comprise an antisense strandwhich corresponds to the antisense strand of an siRNA molecule, e.g.,one of the siRNA molecules described herein. In certain embodiments,aiRNAs of the invention may silence SMAD4 gene expression. In particularembodiments, aiRNAs targeting SMAD4 mRNA are administered using acarrier system such as a nucleic acid-lipid particle. In a preferredembodiment, the nucleic acid-lipid particle comprises: (a) one or moreaiRNA molecules targeting SMAD4 gene expression; (b) a cationic lipid(e.g., DLinDMA, DLenDMA, DLin-K-C2-DMA, and/or γ-DLenDMA); and (c) anon-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol). Incertain instances, the nucleic acid-lipid particle may further comprisea conjugated lipid that prevents aggregation of particles (e.g., PEG-DAAand/or POZ-DAA).

Suitable aiRNA sequences can be identified, synthesized, and modifiedusing any means known in the art for designing, synthesizing, andmodifying siRNA sequences. Additional embodiments related to the aiRNAmolecules of the invention are described in U.S. patent application Ser.No. 12/343,342, filed Dec. 23, 2008, and U.S. patent application Ser.No. 12/424,367, filed Apr. 15, 2009, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

E. miRNA

Generally, microRNAs (miRNA) are single-stranded RNA molecules of about21-23 nucleotides in length which regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed, but miRNAs are nottranslated into protein (non-coding RNA); instead, each primarytranscript (a pri-miRNA) is processed into a short stem-loop structurecalled a pre-miRNA and finally into a functional mature miRNA. MaturemiRNA molecules are either partially or completely complementary to oneor more messenger RNA (mRNA) molecules, and their main function is todownregulate gene expression. The identification of miRNA molecules isdescribed, e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau etal., Science, 294:858-862; and Lee et al., Science, 294:862-864.

The genes encoding miRNA are much longer than the processed mature miRNAmolecule. miRNA are first transcribed as primary transcripts orpri-miRNA with a cap and poly-A tail and processed to short,˜70-nucleotide stem-loop structures known as pre-miRNA in the cellnucleus. This processing is performed in animals by a protein complexknown as the Microprocessor complex, consisting of the nuclease Droshaand the double-stranded RNA binding protein Pasha (Denli et al., Nature,432:231-235 (2004)). These pre-miRNA are then processed to mature miRNAin the cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC)(Bernstein et al., Nature, 409:363-366 (2001). Either the sense strandor antisense strand of DNA can function as templates to give rise tomiRNA.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC complex.This strand is known as the guide strand and is selected by theargonaute protein, the catalytically active RNase in the RISC complex,on the basis of the stability of the 5′ end (Preall et al., Curr. Biol.,16:530-535 (2006)). The remaining strand, known as the anti-guide orpassenger strand, is degraded as a RISC complex substrate (Gregory etal., Cell, 123:631-640 (2005)). After integration into the active RISCcomplex, miRNAs base pair with their complementary mRNA molecules andinduce target mRNA degradation and/or translational silencing.

Mammalian miRNA molecules are usually complementary to a site in the 3′UTR of the target mRNA sequence. In certain instances, the annealing ofthe miRNA to the target mRNA inhibits protein translation by blockingthe protein translation machinery. In certain other instances, theannealing of the miRNA to the target mRNA facilitates the cleavage anddegradation of the target mRNA through a process similar to RNAinterference (RNAi). miRNA may also target methylation of genomic siteswhich correspond to targeted mRNA. Generally, miRNA function inassociation with a complement of proteins collectively termed the miRNP.

In certain aspects, the miRNA molecules described herein are about15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and are preferably about 20-24, 21-22, or 21-23 nucleotides inlength. In certain other aspects, miRNA molecules may comprise one ormore modified nucleotides. As a non-limiting example, miRNA sequencesmay comprise one or more of the modified nucleotides described above forsiRNA sequences. In a preferred embodiment, the miRNA molecule comprises2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, or mixtures thereof.

In some embodiments, miRNA molecules may be used to silence SMAD4 geneexpression. In particular embodiments, miRNAs targeting SMAD4 mRNA areadministered using a carrier system such as a nucleic acid-lipidparticle. In a preferred embodiment, the nucleic acid-lipid particlecomprises: (a) one or more miRNA molecules targeting SMAD4 geneexpression; (b) a cationic lipid (e.g., DLinDMA, DLenDMA, DLin-K-C2-DMA,and/or γ-DLenDMA); and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE,and/or cholesterol). In certain instances, the nucleic acid-lipidparticle may further comprise a conjugated lipid that preventsaggregation of particles (e.g., PEG-DAA and/or POZ-DAA).

In other embodiments, one or more agents that block the activity of anmiRNA targeting SMAD4 mRNA are administered using a lipid particle ofthe invention (e.g., a nucleic acid-lipid particle). Examples ofblocking agents include, but are not limited to, steric blockingoligonucleotides, locked nucleic acid oligonucleotides, and Morpholinooligonucleotides. Such blocking agents may bind directly to the miRNA orto the miRNA binding site on the target RNA.

V. Carrier Systems Containing Therapeutic Nucleic Acids

In one aspect, the present invention provides carrier systems containingone or more therapeutic nucleic acids (e.g., interfering RNA such asdsRNA). In some embodiments, the carrier system is a lipid-based carriersystem such as a lipid particle (e.g., SNALP), a cationic lipid orliposome nucleic acid complex (i.e., lipoplex), a liposome, a micelle, avirosome, or a mixture thereof. In other embodiments, the carrier systemis a polymer-based carrier system such as a cationic polymer-nucleicacid complex (i.e., polyplex). In additional embodiments, the carriersystem is a cyclodextrin-based carrier system such as a cyclodextrinpolymer-nucleic acid complex. In further embodiments, the carrier systemis a protein-based carrier system such as a cationic peptide-nucleicacid complex. Preferably, the carrier system is a lipid particle such asa SNALP. One skilled in the art will appreciate that the therapeuticnucleic acids of the present invention can also be delivered as a nakedmolecule.

A. Lipid Particles

In certain aspects, the present invention provides lipid particlescomprising one or more therapeutic nucleic acids (e.g., interfering RNAsuch as dsRNA) and one or more of cationic (amino) lipids or saltsthereof. In some embodiments, the lipid particles of the inventionfurther comprise one or more non-cationic lipids. In other embodiments,the lipid particles further comprise one or more conjugated lipidscapable of reducing or inhibiting particle aggregation.

The lipid particles of the invention preferably comprise a therapeuticnucleic acid such as an interfering RNA (e.g., siRNA), a cationic lipid,a non-cationic lipid, and a conjugated lipid that inhibits aggregationof particles. In some embodiments, the therapeutic nucleic acid is fullyencapsulated within the lipid portion of the lipid particle such thatthe therapeutic nucleic acid in the lipid particle is resistant inaqueous solution to nuclease degradation. In other embodiments, thelipid particles described herein are substantially non-toxic to mammalssuch as humans. The lipid particles of the invention typically have amean diameter of from about 30 nm to about 150 nm, from about 40 nm toabout 150 nm, from about 50 nm to about 150 nm, from about 60 nm toabout 130 nm, from about 70 nm to about 110 nm, or from about 70 toabout 90 nm. The lipid particles of the invention also typically have alipid:therapeutic agent (e.g., lipid:nucleic acid) ratio (mass/massratio) of from about 1:1 to about 100:1, from about 1:1 to about 50:1,from about 2:1 to about 25:1, from about 3:1 to about 20:1, from about5:1 to about 15:1, or from about 5:1 to about 10:1.

In preferred embodiments, the lipid particles of the invention areserum-stable nucleic acid-lipid particles (SNALP) which comprise aninterfering RNA (e.g., dsRNA such as siRNA, Dicer-substrate dsRNA,shRNA, aiRNA, and/or miRNA), a cationic lipid (e.g., one or morecationic lipids of Formula I-III or salts thereof as set forth herein),a non-cationic lipid (e.g., mixtures of one or more phospholipids andcholesterol), and a conjugated lipid that inhibits aggregation of theparticles (e.g., one or more PEG-lipid conjugates). The SNALP maycomprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodifiedand/or modified interfering RNA (e.g., siRNA) that target one or more ofthe genes described herein. Nucleic acid-lipid particles and theirmethod of preparation are described in, e.g., U.S. Pat. Nos. 5,753,613;5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and 6,320,017;and PCT Publication No. WO 96/40964, the disclosures of which are eachherein incorporated by reference in their entirety for all purposes.

In the nucleic acid-lipid particles of the invention, the nucleic acidmay be fully encapsulated within the lipid portion of the particle,thereby protecting the nucleic acid from nuclease degradation. Inpreferred embodiments, a SNALP comprising a nucleic acid such as aninterfering RNA is fully encapsulated within the lipid portion of theparticle, thereby protecting the nucleic acid from nuclease degradation.In certain instances, the nucleic acid in the SNALP is not substantiallydegraded after exposure of the particle to a nuclease at 37° C. for atleast about 20, 30, 45, or 60 minutes. In certain other instances, thenucleic acid in the SNALP is not substantially degraded after incubationof the particle in serum at 37° C. for at least about 30, 45, or 60minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, thenucleic acid is complexed with the lipid portion of the particle. One ofthe benefits of the formulations of the present invention is that thenucleic acid-lipid particle compositions are substantially non-toxic tomammals such as humans.

The term “fully encapsulated” indicates that the nucleic acid in thenucleic acid-lipid particle is not significantly degraded after exposureto serum or a nuclease assay that would significantly degrade free DNAor RNA. In a fully encapsulated system, preferably less than about 25%of the nucleic acid in the particle is degraded in a treatment thatwould normally degrade 100% of free nucleic acid, more preferably lessthan about 10%, and most preferably less than about 5% of the nucleicacid in the particle is degraded. “Fully encapsulated” also indicatesthat the nucleic acid-lipid particles are serum-stable, that is, thatthey do not rapidly decompose into their component parts upon in vivoadministration.

In the context of nucleic acids, full encapsulation may be determined byperforming a membrane-impermeable fluorescent dye exclusion assay, whichuses a dye that has enhanced fluorescence when associated with nucleicacid. Specific dyes such as OliGreen® and RiboGreen® (Invitrogen Corp.;Carlsbad, Calif.) are available for the quantitative determination ofplasmid DNA, single-stranded deoxyribonucleotides, and/or single- ordouble-stranded ribonucleotides. Encapsulation is determined by addingthe dye to a liposomal formulation, measuring the resultingfluorescence, and comparing it to the fluorescence observed uponaddition of a small amount of nonionic detergent. Detergent-mediateddisruption of the liposomal bilayer releases the encapsulated nucleicacid, allowing it to interact with the membrane-impermeable dye. Nucleicacid encapsulation may be calculated as E=(I_(o)−I)/I_(o), where I andI_(o) refer to the fluorescence intensities before and after theaddition of detergent (see, Wheeler et al., Gene Ther., 6:271-281(1999)).

In other embodiments, the present invention provides a nucleicacid-lipid particle (e.g., SNALP) composition comprising a plurality ofnucleic acid-lipid particles.

In some instances, the SNALP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the particles have the nucleic acid encapsulated therein.

In other instances, the SNALP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the input nucleic acid is encapsulated in the particles.

Depending on the intended use of the lipid particles of the invention,the proportions of the components can be varied and the deliveryefficiency of a particular formulation can be measured using, e.g., anendosomal release parameter (ERP) assay.

1. Cationic Lipids

Any of a variety of cationic lipids or salts thereof may be used in thelipid particles of the present invention (e.g., SNALP), either alone orin combination with one or more other cationic lipid species ornon-cationic lipid species. The cationic lipids include the (R) and/or(S) enantiomers thereof.

In one aspect, cationic lipids of Formula I having the followingstructure are useful in the present invention:

or salts thereof, wherein:

R¹ and R² are either the same or different and are independentlyhydrogen (H) or an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, orC₂-C₆ alkynyl, or R¹ and R² may join to form an optionally substitutedheterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selectedfrom the group consisting of nitrogen (N), oxygen (O), and mixturesthereof;

R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to provide aquaternary amine;

R⁴ and R⁵ are either the same or different and are independently anoptionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl, C₁₀-C₂₄ alkynyl,or C₁₀-C₂₄ acyl, wherein at least one of R⁴ and R⁵ comprises at leasttwo sites of unsaturation; and

n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In onepreferred embodiment, R¹ and R² are both methyl groups. In otherpreferred embodiments, n is 1 or 2. In other embodiments, R³ is absentwhen the pH is above the pK_(a) of the cationic lipid and R³ is hydrogenwhen the pH is below the pK_(a) of the cationic lipid such that theamino head group is protonated. In an alternative embodiment, R³ is anoptionally substituted C₁-C₄ alkyl to provide a quaternary amine 1nfurther embodiments, R⁴ and R⁵ are independently an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl, wherein at leastone of R⁴ and R⁵ comprises at least two sites of unsaturation.

In certain embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, anarachidonyl moiety, and a docosahexaenoyl moiety, as well as acylderivatives thereof (e.g., linoleoyl, linolenoyl, γ-linolenoyl, etc.).In some instances, one of R⁴ and R⁵ comprises a branched alkyl group(e.g., a phytanyl moiety) or an acyl derivative thereof (e.g., aphytanoyl moiety). In certain instances, the octadecadienyl moiety is alinoleyl moiety. In certain other instances, the octadecatrienyl moietyis a linolenyl moiety or a γ-linolenyl moiety. In certain embodiments,R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties, or γ-linolenylmoieties. In particular embodiments, the cationic lipid of Formula I is1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDMA),1,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDAP), ormixtures thereof.

In some embodiments, the cationic lipid of Formula I forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula I is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of cationic lipids such as DLinDMA and DLenDMA, as well asadditional cationic lipids, is described in U.S. Patent Publication No.20060083780, the disclosure of which is herein incorporated by referencein its entirety for all purposes. The synthesis of cationic lipids suchas C2-DLinDMA and C2-DLinDAP, as well as additional cationic lipids, isdescribed in international patent application number WO2011/000106 thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

In another aspect, cationic lipids of Formula II having the followingstructure (or salts thereof) are useful in the present invention:

wherein R¹ and R² are either the same or different and are independentlyan optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄alkynyl, or C₁₂-C₂₄ acyl; R³ and R⁴ are either the same or different andare independently an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,or C₂-C₆ alkynyl, or R³ and R⁴ may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms chosen from nitrogen and oxygen; R⁵ is either absent or ishydrogen (H) or a C₁-C₆ alkyl to provide a quaternary amine; m, n, and pare either the same or different and are independently either 0, 1, or2, with the proviso that m, n, and p are not simultaneously 0; q is 0,1, 2, 3, or 4; and Y and Z are either the same or different and areindependently O, S, or NH. In a preferred embodiment, q is 2.

In some embodiments, the cationic lipid of Formula II is2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2” or “C2K”),2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA;“C3K”), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane(DLin-K-C4-DMA; “C4K”),2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA),2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DO-K-DMA),2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DS-K-DMA),2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane (DLin-K-MA),2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride (DLin-K-TMA.C1), 2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane(DLin-K2-DMA), 2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane(D-Lin-K-N-methylpiperzine), or mixtures thereof. In preferredembodiments, the cationic lipid of Formula II is DLin-K-C2-DMA.

In some embodiments, the cationic lipid of Formula II forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula II is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of cationic lipids such as DLin-K-DMA, as well asadditional cationic lipids, is described in PCT Publication No. WO09/086,558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. The synthesis of cationic lipids suchas DLin-K-C2-DMA, DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ,DO-K-DMA, DS-K-DMA, DLin-K-MA, DLin-K-TMA.Cl, DLin-K²-DMA, andD-Lin-K-N-methylpiperzine, as well as additional cationic lipids, isdescribed in PCT Application No. PCT/US2009/060251, entitled “ImprovedAmino Lipids and Methods for the Delivery of Nucleic Acids,” filed Oct.9, 2009, the disclosure of which is incorporated herein by reference inits entirety for all purposes.

In a further aspect, cationic lipids of Formula III having the followingstructure are useful in the present invention:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either absentor present and when present are either the same or different and areindependently an optionally substituted C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl;and n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, R⁴ and R⁵ are both butyl groups. In yet another preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substituted C₂-C₆or C₂-C₄ alkyl or C₂-C₆ or C₂-C₄ alkenyl.

In an alternative embodiment, the cationic lipid of Formula IIIcomprises ester linkages between the amino head group and one or both ofthe alkyl chains. In some embodiments, the cationic lipid of Formula IIIforms a salt (preferably a crystalline salt) with one or more anions. Inone particular embodiment, the cationic lipid of Formula III is theoxalate (e.g., hemioxalate) salt thereof, which is preferably acrystalline salt.

Although each of the alkyl chains in Formula III contains cis doublebonds at positions 6, 9, and 12 (i.e., cis,cis,cis-Δ⁶,Δ⁹,Δ¹²), in analternative embodiment, one, two, or three of these double bonds in oneor both alkyl chains may be in the trans configuration.

In a particularly preferred embodiment, the cationic lipid of FormulaIII has the structure:

The synthesis of cationic lipids such as γ-DLenDMA, as well asadditional cationic lipids, is described in U.S. Provisional ApplicationNo. 61/222,462, entitled “Improved Cationic Lipids and Methods for theDelivery of Nucleic Acids,” filed Jul. 1, 2009, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

In particular embodiments, a cationic lipid having the followingstructure is useful in the present invention:

The synthesis of cationic lipids such as DLin-M-C3-DMA (“MC3”), as wellas additional cationic lipids (e.g., certain analogs of MC3), isdescribed in U.S. Provisional Application No. 61/185,800, entitled“Novel Lipids and Compositions for the Delivery of Therapeutics,” filedJun. 10, 2009, and U.S. Provisional Application No. 61/287,995, entitled“Methods and Compositions for Delivery of Nucleic Acids,” filed Dec. 18,2009, the disclosures of which are herein incorporated by reference intheir entirety for all purposes.

Examples of other cationic lipids or salts thereof which may be includedin the lipid particles of the present invention include, but are notlimited to, cationic lipids such as those described in WO2011/000106,the disclosure of which is herein incorporated by reference in itsentirety for all purposes, as well as cationic lipids such asN,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-dioeylcarbamoyloxy-3-dimethylaminopropane (DO-C-DAP),1,2-dimyristoleoyl-3-dimethylaminopropane (DMDAP),1,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.Cl),dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA; also known asDLin-M-K-DMA or DLin-M-DMA), and mixtures thereof. Additional cationiclipids or salts thereof which may be included in the lipid particles ofthe present invention are described in U.S. Patent Publication No.20090023673, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The synthesis of cationic lipids such as CLinDMA, as well as additionalcationic lipids, is described in U.S. Patent Publication No.20060240554, the disclosure of which is herein incorporated by referencein its entirety for all purposes. The synthesis of cationic lipids suchas DLin-C-DAP, DLinDAC, DLinMA, DLinDAP, DLin-S-DMA, DLin-2-DMAP,DLinTMA.Cl, DLinTAP.Cl, DLinMPZ, DLinAP, DOAP, and DLin-EG-DMA, as wellas additional cationic lipids, is described in PCT Publication No. WO09/086,558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. The synthesis of cationic lipids suchas DO-C-DAP, DMDAP, DOTAP.Cl, DLin-M-C2-DMA, as well as additionalcationic lipids, is described in PCT Application No. PCT/US2009/060251,entitled “Improved Amino Lipids and Methods for the Delivery of NucleicAcids,” filed Oct. 9, 2009, the disclosure of which is incorporatedherein by reference in its entirety for all purposes. The synthesis of anumber of other cationic lipids and related analogs has been describedin U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613;and 5,785,992; and PCT Publication No. WO 96/10390, the disclosures ofwhich are each herein incorporated by reference in their entirety forall purposes. Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN® (including DOTMAand DOPE, available from Invitrogen); LIPOFECTAMINE® (including DOSPAand DOPE, available from Invitrogen); and TRANSFECTAM® (including DOGS,available from Promega Corp.).

In some embodiments, the cationic lipid comprises from about 50 mol % toabout 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol% to about 80 mol %, from about 50 mol % to about 75 mol %, from about50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, fromabout 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %,or from about 55 mol % to about 70 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle. In particularembodiments, the cationic lipid comprises about 50 mol %, 51 mol %, 52mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59mol %, 60 mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (orany fraction thereof) of the total lipid present in the particle.

In other embodiments, the cationic lipid comprises from about 2 mol % toabout 60 mol %, from about 5 mol % to about 50 mol %, from about 10 mol% to about 50 mol %, from about 20 mol % to about 50 mol %, from about20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %, orabout 40 mol % (or any fraction thereof or range therein) of the totallipid present in the particle.

Additional percentages and ranges of cationic lipids suitable for use inthe lipid particles of the present invention are described in PCTPublication No. WO 09/127060, U.S. Published Application No. US2011/0071208, PCT Publication No. WO2011/000106, and U.S. PublishedApplication No. US 2011/0076335, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of cationic lipid present inthe lipid particles of the invention is a target amount, and that theactual amount of cationic lipid present in the formulation may vary, forexample, by ±5 mol %. For example, in the 1:57 lipid particle (e.g.,SNALP) formulation, the target amount of cationic lipid is 57.1 mol %,but the actual amount of cationic lipid may be ±5 mol %, ±4 mol %, ±3mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1mol % of that target amount, with the balance of the formulation beingmade up of other lipid components (adding up to 100 mol % of totallipids present in the particle).

2. Non-Cationic Lipids

The non-cationic lipids used in the lipid particles of the invention(e.g., SNALP) can be any of a variety of neutral uncharged,zwitterionic, or anionic lipids capable of producing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C10-C24 carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof. Non-limiting examples ofcholesterol derivatives include polar analogues such as 5α-cholestanol,5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether,cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polaranalogues such as 5α-cholestane, cholestenone, 5α-cholestanone,5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. Inpreferred embodiments, the cholesterol derivative is a polar analoguesuch as cholesteryl-(4′-hydroxy)-butyl ether. The synthesis ofcholesteryl-(2′-hydroxy)-ethyl ether is described in PCT Publication No.WO 09/127060, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

In some embodiments, the non-cationic lipid present in the lipidparticles (e.g., SNALP) comprises or consists of a mixture of one ormore phospholipids and cholesterol or a derivative thereof. In otherembodiments, the non-cationic lipid present in the lipid particles(e.g., SNALP) comprises or consists of one or more phospholipids, e.g.,a cholesterol-free lipid particle formulation. In yet other embodiments,the non-cationic lipid present in the lipid particles (e.g., SNALP)comprises or consists of cholesterol or a derivative thereof, e.g., aphospholipid-free lipid particle formulation.

Other examples of non-cationic lipids suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

In some embodiments, the non-cationic lipid comprises from about 10 mol% to about 60 mol %, from about 20 mol % to about 55 mol %, from about20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, fromabout 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %,from about 30 mol % to about 50 mol %, from about 30 mol % to about 45mol %, from about 30 mol % to about 40 mol %, from about 35 mol % toabout 45 mol %, from about 37 mol % to about 42 mol %, or about 35 mol%, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %,43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In embodiments where the lipid particles contain a mixture ofphospholipid and cholesterol or a cholesterol derivative, the mixturemay comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60mol % of the total lipid present in the particle.

In some embodiments, the phospholipid component in the mixture maycomprise from about 2 mol % to about 20 mol %, from about 2 mol % toabout 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol %to about 15 mol %, or from about 4 mol % to about 10 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the phospholipid componentin the mixture comprises from about 5 mol % to about 10 mol %, fromabout 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %,from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol%, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. As a non-limiting example, a 1:57 lipid particle formulationcomprising a mixture of phospholipid and cholesterol may comprise aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof), e.g., in a mixture with cholesterol or a cholesterolderivative at about 34 mol % (or any fraction thereof) of the totallipid present in the particle. As another non-limiting example, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise a phospholipid such as DPPC or DSPC at about 7mol % (or any fraction thereof), e.g., in a mixture with cholesterol ora cholesterol derivative at about 32 mol % (or any fraction thereof) ofthe total lipid present in the particle.

In other embodiments, the cholesterol component in the mixture maycomprise from about 25 mol % to about 45 mol %, from about 25 mol % toabout 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol% to about 40 mol %, from about 27 mol % to about 37 mol %, from about25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the cholesterol component inthe mixture comprises from about 25 mol % to about 35 mol %, from about27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, fromabout 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %,from about 31 mol % to about 33 mol %, or about 30 mol %, 31 mol %, 32mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle. Typically, a 1:57lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise cholesterol or a cholesterol derivative atabout 34 mol % (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle. Typically, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise cholesterol or a cholesterol derivative atabout 32 mol % (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle.

In embodiments where the lipid particles are phospholipid-free, thecholesterol or derivative thereof may comprise up to about 25 mol %, 30mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % ofthe total lipid present in the particle.

In some embodiments, the cholesterol or derivative thereof in thephospholipid-free lipid particle formulation may comprise from about 25mol % to about 45 mol %, from about 25 mol % to about 40 mol %, fromabout 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %,from about 31 mol % to about 39 mol %, from about 32 mol % to about 38mol %, from about 33 mol % to about 37 mol %, from about 35 mol % toabout 45 mol %, from about 30 mol % to about 35 mol %, from about 35 mol% to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. As a non-limiting example, a 1:62 lipid particle formulationmay comprise cholesterol at about 37 mol % (or any fraction thereof) ofthe total lipid present in the particle. As another non-limitingexample, a 7:58 lipid particle formulation may comprise cholesterol atabout 35 mol % (or any fraction thereof) of the total lipid present inthe particle.

In other embodiments, the non-cationic lipid comprises from about 5 mol% to about 90 mol %, from about 10 mol % to about 85 mol %, from about20 mol % to about 80 mol %, about 10 mol % (e.g., phospholipid only), orabout 60 mol % (e.g., phospholipid and cholesterol or derivativethereof) (or any fraction thereof or range therein) of the total lipidpresent in the particle.

Additional percentages and ranges of non-cationic lipids suitable foruse in the lipid particles of the present invention are described in PCTPublication No. WO 09/127060, U.S. Published Application No. US2011/0071208, PCT Publication No. WO2011/000106, and U.S. PublishedApplication No. US 2011/0076335, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of non-cationic lipidpresent in the lipid particles of the invention is a target amount, andthat the actual amount of non-cationic lipid present in the formulationmay vary, for example, by ±5 mol %. For example, in the 1:57 lipidparticle (e.g., SNALP) formulation, the target amount of phospholipid is7.1 mol % and the target amount of cholesterol is 34.3 mol %, but theactual amount of phospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that targetamount, and the actual amount of cholesterol may be ±3 mol %, ±2 mol %,±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle). Similarly, in the 7:54 lipid particle (e.g., SNALP)formulation, the target amount of phospholipid is 6.75 mol % and thetarget amount of cholesterol is 32.43 mol %, but the actual amount ofphospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %, ±0.75 mol %, ±0.5mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, and the actualamount of cholesterol may be ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, with thebalance of the formulation being made up of other lipid components(adding up to 100 mol % of total lipids present in the particle).

3. Lipid Conjugates

In addition to cationic and non-cationic lipids, the lipid particles ofthe invention (e.g., SNALP) may further comprise a lipid conjugate. Theconjugated lipid is useful in that it prevents the aggregation ofparticles. Suitable conjugated lipids include, but are not limited to,PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates,cationic-polymer-lipid conjugates (CPLs), and mixtures thereof. Incertain embodiments, the particles comprise either a PEG-lipid conjugateor an ATTA-lipid conjugate together with a CPL.

In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examplesof PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEGcoupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613,PEG conjugated to cholesterol or a derivative thereof, and mixturesthereof. The disclosures of these patent documents are hereinincorporated by reference in their entirety for all purposes.

Additional PEG-lipids suitable for use in the invention include, withoutlimitation, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG).The synthesis of PEG-C-DOMG is described in PCT Publication No. WO09/086,558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. Yet additional suitable PEG-lipidconjugates include, without limitation,1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (2 KPEG-DMG). The synthesis of 2 KPEG-DMG is described in U.S.Pat. No. 7,404,969, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, but are not limited to, thefollowing: monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S—NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES),monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as wellas such compounds containing a terminal hydroxyl group instead of aterminal methoxy group (e.g., HO-PEG-S, HO-PEG-S—NHS, HO-PEG-NH2, etc.).Other PEGs such as those described in U.S. Pat. Nos. 6,774,180 and7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing thePEG-lipid conjugates of the present invention. The disclosures of thesepatents are herein incorporated by reference in their entirety for allpurposes. In addition, monomethoxypolyethyleneglycol-acetic acid(MePEG-CH₂COOH) is particularly useful for preparing PEG-lipidconjugates including, e.g., PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). Inpreferred embodiments, the PEG moiety has an average molecular weight ofabout 2,000 daltons or about 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skilled in the art.Phosphatidyl-ethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C10 to C20 arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

The term “ATTA” or “polyamide” includes, without limitation, compoundsdescribed in U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. These compounds include a compound having the formula:

wherein R is a member selected from the group consisting of hydrogen,alkyl and acyl; R1 is a member selected from the group consisting ofhydrogen and alkyl; or optionally, R and R1 and the nitrogen to whichthey are bound form an azido moiety; R2 is a member of the groupselected from hydrogen, optionally substituted alkyl, optionallysubstituted aryl and a side chain of an amino acid; R³ is a memberselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, mercapto, hydrazino, amino and NR4R5, wherein R4 and R5 areindependently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4;and q is 0 or 1. It will be apparent to those of skill in the art thatother polyamides can be used in the compounds of the present invention.

The term “diacylglycerol” or “DAG” includes a compound having 2 fattyacyl chains, R¹ and R², both of which have independently between 2 and30 carbons bonded to the 1- and 2-position of glycerol by esterlinkages. The acyl groups can be saturated or have varying degrees ofunsaturation. Suitable acyl groups include, but are not limited to,lauroyl (C₁₂), myristoyl (C₁₄), palmitoyl (C₁₆), stearoyl (C₁₈), andicosoyl (C₂₀). In preferred embodiments, R¹ and R² are the same, i.e.,R¹ and R² are both myristoyl (i.e., dimyristoyl), R¹ and R² are bothstearoyl (i.e., distearoyl), etc. Diacylglycerols have the followinggeneral formula:

The term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkylchains, R1 and R2, both of which have independently between 2 and 30carbons. The alkyl groups can be saturated or have varying degrees ofunsaturation. Dialkyloxypropyls have the following general formula:

In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate havingthe following formula:

wherein R¹ and R² are independently selected and are long-chain alkylgroups having from about 10 to about 22 carbon atoms; PEG is apolyethyleneglycol; and L is a non-ester containing linker moiety or anester containing linker moiety as described above. The long-chain alkylgroups can be saturated or unsaturated. Suitable alkyl groups include,but are not limited to, decyl (C₁₀), lauryl (C₁₂), myristyl (C₁₄),palmityl (C₁₆), stearyl (C₁₈), and icosyl (C₂₀). In preferredembodiments, R¹ and R² are the same, i.e., R¹ and R² are both myristyl(i.e., dimyristyl), R¹ and R² are both stearyl (i.e., distearyl), etc.

In Formula VII above, the PEG has an average molecular weight rangingfrom about 550 daltons to about 10,000 daltons. In certain instances,the PEG has an average molecular weight of from about 750 daltons toabout 5,000 daltons (e.g., from about 1,000 daltons to about 5,000daltons, from about 1,500 daltons to about 3,000 daltons, from about 750daltons to about 3,000 daltons, from about 750 daltons to about 2,000daltons, etc.). In preferred embodiments, the PEG has an averagemolecular weight of about 2,000 daltons or about 750 daltons. The PEGcan be optionally substituted with alkyl, alkoxy, acyl, or aryl groups.In certain embodiments, the terminal hydroxyl group is substituted witha methoxy or methyl group.

In a preferred embodiment, “L” is a non-ester containing linker moiety.Suitable non-ester containing linkers include, but are not limited to,an amido linker moiety, an amino linker moiety, a carbonyl linkermoiety, a carbamate linker moiety, a urea linker moiety, an ether linkermoiety, a disulphide linker moiety, a succinamidyl linker moiety, andcombinations thereof. In a preferred embodiment, the non-estercontaining linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAAconjugate). In another preferred embodiment, the non-ester containinglinker moiety is an amido linker moiety (i.e., a PEG-A-DAA conjugate).In yet another preferred embodiment, the non-ester containing linkermoiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).

In particular embodiments, the PEG-lipid conjugate is selected from:

The PEG-DAA conjugates are synthesized using standard techniques andreagents known to those of skill in the art. It will be recognized thatthe PEG-DAA conjugates will contain various amide, amine, ether, thio,carbamate, and urea linkages. Those of skill in the art will recognizethat methods and reagents for forming these bonds are well known andreadily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); andFurniss, VOGEL′S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed.(Longman 1989). It will also be appreciated that any functional groupspresent may require protection and deprotection at different points inthe synthesis of the PEG-DAA conjugates. Those of skill in the art willrecognize that such techniques are well known. See, e.g., Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C10)conjugate, a PEG-dilauryloxypropyl (C12) conjugate, aPEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C₁₆)conjugate, or a PEG-distearyloxypropyl (C₁₈) conjugate. In theseembodiments, the PEG preferably has an average molecular weight of about750 or about 2,000 daltons. In one particularly preferred embodiment,the PEG-lipid conjugate comprises PEG2000-C-DMA, wherein the “2000”denotes the average molecular weight of the PEG, the “C” denotes acarbamate linker moiety, and the “DMA” denotes dimyristyloxypropyl. Inanother particularly preferred embodiment, the PEG-lipid conjugatecomprises PEG750-C-DMA, wherein the “750” denotes the average molecularweight of the PEG, the “C” denotes a carbamate linker moiety, and the“DMA” denotes dimyristyloxypropyl. In particular embodiments, theterminal hydroxyl group of the PEG is substituted with a methyl group.Those of skill in the art will readily appreciate that otherdialkyloxypropyls can be used in the PEG-DAA conjugates of the presentinvention.

In addition to the foregoing, it will be readily apparent to those ofskill in the art that other hydrophilic polymers can be used in place ofPEG. Examples of suitable polymers that can be used in place of PEGinclude, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

In addition to the foregoing components, the lipid particles (e.g.,SNALP) of the present invention can further comprise cationicpoly(ethylene glycol) (PEG) lipids or CPLs (see, e.g., Chen et al.,Bioconj. Chem., 11:433-437 (2000); U.S. Pat. No. 6,852,334; PCTPublication No. WO 00/62813, the disclosures of which are hereinincorporated by reference in their entirety for all purposes).

Suitable CPLs include compounds of Formula VIII:A-W-Y  (VIII),wherein A, W, and Y are as described below.

With reference to Formula VIII, “A” is a lipid moiety such as anamphipathic lipid, a neutral lipid, or a hydrophobic lipid that acts asa lipid anchor. Suitable lipid examples include, but are not limited to,diacylglycerolyls, dialkylglycerolyls, N—N-dialkylaminos,1,2-diacyloxy-3-aminopropanes, and 1,2-dialkyl-3-aminopropanes.

“W” is a polymer or an oligomer such as a hydrophilic polymer oroligomer. Preferably, the hydrophilic polymer is a biocompatable polymerthat is nonimmunogenic or possesses low inherent immunogenicity.Alternatively, the hydrophilic polymer can be weakly antigenic if usedwith appropriate adjuvants. Suitable nonimmunogenic polymers include,but are not limited to, PEG, polyamides, polylactic acid, polyglycolicacid, polylactic acid/polyglycolic acid copolymers, and combinationsthereof. In a preferred embodiment, the polymer has a molecular weightof from about 250 to about 7,000 daltons.

“Y” is a polycationic moiety. The term polycationic moiety refers to acompound, derivative, or functional group having a positive charge,preferably at least 2 positive charges at a selected pH, preferablyphysiological pH. Suitable polycationic moieties include basic aminoacids and their derivatives such as arginine, asparagine, glutamine,lysine, and histidine; spermine; spermidine; cationic dendrimers;polyamines; polyamine sugars; and amino polysaccharides. Thepolycationic moieties can be linear, such as linear tetralysine,branched or dendrimeric in structure. Polycationic moieties have betweenabout 2 to about 15 positive charges, preferably between about 2 toabout 12 positive charges, and more preferably between about 2 to about8 positive charges at selected pH values. The selection of whichpolycationic moiety to employ may be determined by the type of particleapplication which is desired.

The charges on the polycationic moieties can be either distributedaround the entire particle moiety, or alternatively, they can be adiscrete concentration of charge density in one particular area of theparticle moiety e.g., a charge spike. If the charge density isdistributed on the particle, the charge density can be equallydistributed or unequally distributed. All variations of chargedistribution of the polycationic moiety are encompassed by the presentinvention.

The lipid “A” and the nonimmunogenic polymer “W” can be attached byvarious methods and preferably by covalent attachment. Methods known tothose of skill in the art can be used for the covalent attachment of “A”and “W.” Suitable linkages include, but are not limited to, amide,amine, carboxyl, carbonate, carbamate, ester, and hydrazone linkages. Itwill be apparent to those skilled in the art that “A” and “W” must havecomplementary functional groups to effectuate the linkage. The reactionof these two groups, one on the lipid and the other on the polymer, willprovide the desired linkage. For example, when the lipid is adiacylglycerol and the terminal hydroxyl is activated, for instance withNHS and DCC, to form an active ester, and is then reacted with a polymerwhich contains an amino group, such as with a polyamide (see, e.g., U.S.Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which are hereinincorporated by reference in their entirety for all purposes), an amidebond will form between the two groups.

In certain instances, the polycationic moiety can have a ligandattached, such as a targeting ligand or a chelating moiety forcomplexing calcium. Preferably, after the ligand is attached, thecationic moiety maintains a positive charge. In certain instances, theligand that is attached has a positive charge. Suitable ligands include,but are not limited to, a compound or device with a reactive functionalgroup and include lipids, amphipathic lipids, carrier compounds,bioaffinity compounds, biomaterials, biopolymers, biomedical devices,analytically detectable compounds, therapeutically active compounds,enzymes, peptides, proteins, antibodies, immune stimulators,radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA,polysaccharides, liposomes, virosomes, micelles, immunoglobulins,functional groups, other targeting moieties, or toxins.

In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % toabout 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8mol % to about 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, fromabout 0.9 mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol%, from about 1 mol % to about 1.7 mol %, from about 1.2 mol % to about1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol %to about 1.6 mol %, or from about 1.4 mol % to about 1.5 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle.

In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % toabout 18 mol %, from about 2 mol % to about 15 mol %, from about 4 mol %to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5mol % to about 12 mol %, or about 2 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle.

In further embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol%, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol% (or any fraction thereof or range therein) of the total lipid presentin the particle.

Additional percentages and ranges of lipid conjugates suitable for usein the lipid particles of the present invention are described in PCTPublication No. WO 09/127,060, U.S. Published Application No. US2011/0071208, PCT Publication No. WO2011/000106, and U.S. PublishedApplication No. US 2011/0076335, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of lipid conjugate (e.g.,PEG-lipid) present in the lipid particles of the invention is a targetamount, and that the actual amount of lipid conjugate present in theformulation may vary, for example, by ±2 mol %. For example, in the 1:57lipid particle (e.g., SNALP) formulation, the target amount of lipidconjugate is 1.4 mol %, but the actual amount of lipid conjugate may be±0.5 mol %, ±0.4 mol %, ±0.3 mol %, ±0.2 mol %, ±0.1 mol %, or ±0.05 mol% of that target amount, with the balance of the formulation being madeup of other lipid components (adding up to 100 mol % of total lipidspresent in the particle). Similarly, in the 7:54 lipid particle (e.g.,SNALP) formulation, the target amount of lipid conjugate is 6.76 mol %,but the actual amount of lipid conjugate may be ±2 mol %, ±1.5 mol %, ±1mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle).

One of ordinary skill in the art will appreciate that the concentrationof the lipid conjugate can be varied depending on the lipid conjugateemployed and the rate at which the lipid particle is to becomefusogenic.

By controlling the composition and concentration of the lipid conjugate,one can control the rate at which the lipid conjugate exchanges out ofthe lipid particle and, in turn, the rate at which the lipid particlebecomes fusogenic. For instance, when a PEG-DAA conjugate is used as thelipid conjugate, the rate at which the lipid particle becomes fusogeniccan be varied, for example, by varying the concentration of the lipidconjugate, by varying the molecular weight of the PEG, or by varying thechain length and degree of saturation of the alkyl groups on the PEG-DAAconjugate. In addition, other variables including, for example, pH,temperature, ionic strength, etc. can be used to vary and/or control therate at which the lipid particle becomes fusogenic. Other methods whichcan be used to control the rate at which the lipid particle becomesfusogenic will become apparent to those of skill in the art upon readingthis disclosure. Also, by controlling the composition and concentrationof the lipid conjugate, one can control the lipid particle (e.g., SNALP)size.

B. Additional Carrier Systems

[Non-limiting examples of additional lipid-based carrier systemssuitable for use in the present invention include lipoplexes (see, e.g.,U.S. Patent Publication No. 20030203865; and Zhang et al., J. ControlRelease, 100:165-180 (2004)), pH-sensitive lipoplexes (see, e.g., U.S.Patent Publication No. 20020192275), reversibly masked lipoplexes (see,e.g., U.S. Patent Publication Nos. 20030180950), cationic lipid-basedcompositions (see, e.g., U.S. Pat. No. 6,756,054; and U.S. PatentPublication No. 20050234232), cationic liposomes (see, e.g., U.S. PatentPublication Nos. 20030229040, 20020160038, and 20020012998; U.S. Pat.No. 5,908,635; and PCT Publication No. WO 01/72283), anionic liposomes(see, e.g., U.S. Patent Publication No. 20030026831), pH-sensitiveliposomes (see, e.g., U.S. Patent Publication No. 20020192274; and AU2003210303), antibody-coated liposomes (see, e.g., U.S. PatentPublication No. 20030108597; and PCT Publication No. WO 00/50008),cell-type specific liposomes (see, e.g., U.S. Patent Publication No.20030198664), liposomes containing nucleic acid and peptides (see, e.g.,U.S. Pat. No. 6,207,456), liposomes containing lipids derivatized withreleasable hydrophilic polymers (see, e.g., U.S. Patent Publication No.20030031704), lipid-entrapped nucleic acid (see, e.g., PCT PublicationNos. WO 03/057190 and WO 03/059322), lipid-encapsulated nucleic acid(see, e.g., U.S. Patent Publication No. 20030129221; and U.S. Pat. No.5,756,122), other liposomal compositions (see, e.g., U.S. PatentPublication Nos. 20030035829 and 20030072794; and U.S. Pat. No.6,200,599), stabilized mixtures of liposomes and emulsions (see, e.g.,EP1304160), emulsion compositions (see, e.g., U.S. Pat. No. 6,747,014),and nucleic acid micro-emulsions (see, e.g., U.S. Patent Publication No.20050037086).

Examples of polymer-based carrier systems suitable for use in thepresent invention include, but are not limited to, cationicpolymer-nucleic acid complexes (i.e., polyplexes). To form a polyplex, anucleic acid (e.g., interfering RNA) is typically complexed with acationic polymer having a linear, branched, star, or dendritic polymericstructure that condenses the nucleic acid into positively chargedparticles capable of interacting with anionic proteoglycans at the cellsurface and entering cells by endocytosis. In some embodiments, thepolyplex comprises nucleic acid (e.g., interfering RNA) complexed with acationic polymer such as polyethylenimine (PEI) (see, e.g., U.S. Pat.No. 6,013,240; commercially available from Qbiogene, Inc. (Carlsbad,Calif.) as In vivo jetPEI™, a linear form of PEI), polypropylenimine(PPI), polyvinylpyrrolidone (PVP), poly-L-lysine (PLL),diethylaminoethyl (DEAE)-dextran, poly(β-amino ester) (PAE) polymers(see, e.g., Lynn et al., J. Am. Chem. Soc., 123:8155-8156 (2001)),chitosan, polyamidoamine (PAMAM) dendrimers (see, e.g., Kukowska-Latalloet al., Proc. Natl. Acad. Sci. USA, 93:4897-4902 (1996)), porphyrin(see, e.g., U.S. Pat. No. 6,620,805), polyvinylether (see, e.g., U.S.Patent Publication No. 20040156909), polycyclic amidinium (see, e.g.,U.S. Patent Publication No. 20030220289), other polymers comprisingprimary amine, imine, guanidine, and/or imidazole groups (see, e.g.,U.S. Pat. No. 6,013,240; PCT Publication No. WO/9602655; PCT PublicationNo. WO95/21931; Zhang et al., J. Control Release, 100:165-180 (2004);and Tiera et al., Curr. Gene Ther., 6:59-71 (2006)), and a mixturethereof. In other embodiments, the polyplex comprises cationicpolymer-nucleic acid complexes as described in U.S. Patent PublicationNos. 20060211643, 20050222064, 20030125281, and 20030185890, and PCTPublication No. WO 03/066069; biodegradable poly(β-amino ester)polymer-nucleic acid complexes as described in U.S. Patent PublicationNo. 20040071654; microparticles containing polymeric matrices asdescribed in U.S. Patent Publication No. 20040142475; othermicroparticle compositions as described in U.S. Patent Publication No.20030157030; condensed nucleic acid complexes as described in U.S.Patent Publication No. 20050123600; and nanocapsule and microcapsulecompositions as described in AU 2002358514 and PCT Publication No. WO02/096551.

In certain instances, the interfering RNA may be complexed withcyclodextrin or a polymer thereof. Non-limiting examples ofcyclodextrin-based carrier systems include the cyclodextrin-modifiedpolymer-nucleic acid complexes described in U.S. Patent Publication No.20040087024; the linear cyclodextrin copolymer-nucleic acid complexesdescribed in U.S. Pat. Nos. 6,509,323, 6,884,789, and 7,091,192; and thecyclodextrin polymer-complexing agent-nucleic acid complexes describedin U.S. Pat. No. 7,018,609. In certain other instances, the interferingRNA may be complexed with a peptide or polypeptide. An example of aprotein-based carrier system includes, but is not limited to, thecationic oligopeptide-nucleic acid complex described in PCT PublicationNo. WO95/21931.

VI. Preparation of Lipid Particles

The lipid particles of the present invention, e.g., SNALP, in which anucleic acid such as an interfering RNA (e.g., siRNA) is entrappedwithin the lipid portion of the particle and is protected fromdegradation, can be formed by any method known in the art including, butnot limited to, a continuous mixing method, a direct dilution process,and an in-line dilution process.

In particular embodiments, the cationic lipids may comprise lipids ofFormula I-III or salts thereof, alone or in combination with othercationic lipids. In other embodiments, the non-cationic lipids are eggsphingomyelin (ESM), distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC),1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC),dipalmitoyl-phosphatidylcholine (DPPC),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,14:0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE(1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE(1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE(1,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1 trans PE(1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE(1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethyleneglycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modifieddiacylglycerols, or PEG-modified dialkyloxypropyls), cholesterol,derivatives thereof, or combinations thereof.

In certain embodiments, the present invention provides nucleicacid-lipid particles (e.g., SNALP) produced via a continuous mixingmethod, e.g., a process that includes providing an aqueous solutioncomprising a nucleic acid (e.g., interfering RNA) in a first reservoir,providing an organic lipid solution in a second reservoir (wherein thelipids present in the organic lipid solution are solubilized in anorganic solvent, e.g., a lower alkanol such as ethanol), and mixing theaqueous solution with the organic lipid solution such that the organiclipid solution mixes with the aqueous solution so as to substantiallyinstantaneously produce a lipid vesicle (e.g., liposome) encapsulatingthe nucleic acid within the lipid vesicle. This process and theapparatus for carrying out this process are described in detail in U.S.Patent Publication No. 20040142025, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The action of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a lipid vesicle substantially instantaneously upon mixing. Asused herein, the phrase “continuously diluting a lipid solution with abuffer solution” (and variations) generally means that the lipidsolution is diluted sufficiently rapidly in a hydration process withsufficient force to effectuate vesicle generation. By mixing the aqueoussolution comprising a nucleic acid with the organic lipid solution, theorganic lipid solution undergoes a continuous stepwise dilution in thepresence of the buffer solution (i.e., aqueous solution) to produce anucleic acid-lipid particle.

The nucleic acid-lipid particles formed using the continuous mixingmethod typically have a size of from about 30 nm to about 150 nm, fromabout 40 nm to about 150 nm, from about 50 nm to about 150 nm, fromabout 60 nm to about 130 nm, from about 70 nm to about 110 nm, fromabout 70 nm to about 100 nm, from about 80 nm to about 100 nm, fromabout 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, or 150 nm (or any fraction thereof or range therein). Theparticles thus formed do not aggregate and are optionally sized toachieve a uniform particle size.

In another embodiment, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) produced via a direct dilution process thatincludes forming a lipid vesicle (e.g., liposome) solution andimmediately and directly introducing the lipid vesicle solution into acollection vessel containing a controlled amount of dilution buffer. Inpreferred aspects, the collection vessel includes one or more elementsconfigured to stir the contents of the collection vessel to facilitatedilution. In one aspect, the amount of dilution buffer present in thecollection vessel is substantially equal to the volume of lipid vesiclesolution introduced thereto. As a non-limiting example, a lipid vesiclesolution in 45% ethanol when introduced into the collection vesselcontaining an equal volume of dilution buffer will advantageously yieldsmaller particles.

In yet another embodiment, the present invention provides nucleicacid-lipid particles (e.g., SNALP) produced via an in-line dilutionprocess in which a third reservoir containing dilution buffer is fluidlycoupled to a second mixing region. In this embodiment, the lipid vesicle(e.g., liposome) solution formed in a first mixing region is immediatelyand directly mixed with dilution buffer in the second mixing region. Inpreferred aspects, the second mixing region includes a T-connectorarranged so that the lipid vesicle solution and the dilution bufferflows meet as opposing 180° flows; however, connectors providingshallower angles can be used, e.g., from about 27° to about 180° (e.g.,about 90°). A pump mechanism delivers a controllable flow of buffer tothe second mixing region. In one aspect, the flow rate of dilutionbuffer provided to the second mixing region is controlled to besubstantially equal to the flow rate of lipid vesicle solutionintroduced thereto from the first mixing region. This embodimentadvantageously allows for more control of the flow of dilution buffermixing with the lipid vesicle solution in the second mixing region, andtherefore also the concentration of lipid vesicle solution in bufferthroughout the second mixing process. Such control of the dilutionbuffer flow rate advantageously allows for small particle size formationat reduced concentrations.

These processes and the apparatuses for carrying out these directdilution and in-line dilution processes are described in detail in U.S.Patent Publication No. 20070042031, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The nucleic acid-lipid particles formed using the direct dilution andin-line dilution processes typically have a size of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, from about 70 nm to about 100 nm, from about 80 nm toabout 100 nm, from about 90 nm to about 100 nm, from about 70 to about90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm,less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm,35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130nm, 135 nm, 140 nm, 145 nm, or 150 nm (or any fraction thereof or rangetherein). The particles thus formed do not aggregate and are optionallysized to achieve a uniform particle size.

If needed, the lipid particles of the invention (e.g., SNALP) can besized by any of the methods available for sizing liposomes. The sizingmay be conducted in order to achieve a desired size range and relativelynarrow distribution of particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles, is described in U.S. Pat. No. 4,737,323, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. Sonicating a particle suspension either by bath orprobe sonication produces a progressive size reduction down to particlesof less than about 50 nm in size. Homogenization is another method whichrelies on shearing energy to fragment larger particles into smallerones. In a typical homogenization procedure, particles are recirculatedthrough a standard emulsion homogenizer until selected particle sizes,typically between about 60 and about 80 nm, are observed. In bothmethods, the particle size distribution can be monitored by conventionallaser-beam particle size discrimination, or QELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In some embodiments, the nucleic acids present in the particles areprecondensed as described in, e.g., U.S. patent application Ser. No.09/744,103, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

In other embodiments, the methods may further comprise adding non-lipidpolycations which are useful to effect the lipofection of cells usingthe present compositions. Examples of suitable non-lipid polycationsinclude, hexadimethrine bromide (sold under the brand name POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine, and polyethyleneimine. Addition of these salts ispreferably after the particles have been formed.

In some embodiments, the nucleic acid to lipid ratios (mass/mass ratios)in a formed nucleic acid-lipid particle (e.g., SNALP) will range fromabout 0.01 to about 0.2, from about 0.05 to about 0.2, from about 0.02to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about0.08. The ratio of the starting materials (input) also falls within thisrange. In other embodiments, the particle preparation uses about 400 μgnucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratioof about 0.01 to about 0.08 and, more preferably, about 0.04, whichcorresponds to 1.25 mg of total lipid per 50 μg of nucleic acid. Inother preferred embodiments, the particle has a nucleic acid:lipid massratio of about 0.08.

In other embodiments, the lipid to nucleic acid ratios (mass/massratios) in a formed nucleic acid-lipid particle (e.g., SNALP) will rangefrom about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100(100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) toabout 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4(4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), fromabout 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1),from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25(25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) toabout 20 (20:1), from about 5 (5:1) to about 15 (15:1), from about 5(5:1) to about 10 (10:1), or about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9(9:1), 10 (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), 15 (15:1),16 (16:1), 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21 (21:1), 22(22:1), 23 (23:1), 24 (24:1), or 25 (25:1), or any fraction thereof orrange therein. The ratio of the starting materials (input) also fallswithin this range.

As previously discussed, the conjugated lipid may further include a CPL.A variety of general methods for making SNALP-CPLs (CPL-containingSNALP) are discussed herein. Two general techniques include the“post-insertion” technique, that is, insertion of a CPL into, forexample, a pre-formed SNALP, and the “standard” technique, wherein theCPL is included in the lipid mixture during, for example, the SNALPformation steps. The post-insertion technique results in SNALP havingCPLs mainly in the external face of the SNALP bilayer membrane, whereasstandard techniques provide SNALP having CPLs on both internal andexternal faces. The method is especially useful for vesicles made fromphospholipids (which can contain cholesterol) and also for vesiclescontaining PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of makingSNALP-CPLs are taught, for example, in U.S. Pat. Nos. 5,705,385;6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent PublicationNo. 20020072121; and PCT Publication No. WO 00/62813, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

VII. Kits

The present invention also provides lipid particles (e.g., SNALP) in kitform. In some embodiments, the kit comprises a container which iscompartmentalized for holding the various elements of the lipidparticles (e.g., the active agents or therapeutic agents such as nucleicacids and the individual lipid components of the particles). Preferably,the kit comprises a container (e.g., a vial or ampoule) which holds thelipid particles of the invention (e.g., SNALP), wherein the particlesare produced by one of the processes set forth herein. In certainembodiments, the kit may further comprise an endosomal membranedestabilizer (e.g., calcium ions). The kit typically contains theparticle compositions of the invention, either as a suspension in apharmaceutically acceptable carrier or in dehydrated form, withinstructions for their rehydration (if lyophilized) and administration.

The SNALP formulations of the present invention can be tailored topreferentially target particular cells, tissues, or organs of interest.Preferential targeting of SNALP may be carried out by controlling thecomposition of the SNALP itself. In particular embodiments, the kits ofthe invention comprise these lipid particles, wherein the particles arepresent in a container as a suspension or in dehydrated form.

In certain instances, it may be desirable to have a targeting moietyattached to the surface of the lipid particle to further enhance thetargeting of the particle. Methods of attaching targeting moieties(e.g., antibodies, proteins, etc.) to lipids (such as those used in thepresent particles) are known to those of skill in the art.

VIII. Administration of Lipid Particles

Once formed, the lipid particles of the invention (e.g., SNALP) areparticularly useful for the introduction of nucleic acids (e.g.,interfering RNA such as dsRNA) into cells. Accordingly, the presentinvention also provides methods for introducing a nucleic acid (e.g.,interfering RNA) into a cell. In particular embodiments, the nucleicacid (e.g., interfering RNA) is introduced into an infected cell such asreticuloendothelial cells (e.g., macrophages, monocytes, etc.) as wellas other cell types, including fibroblasts, endothelial cells (such asthose lining the interior surface of blood vessels), and/or plateletcells. The methods may be carried out in vitro or in vivo by firstforming the particles as described above and then contacting theparticles with the cells for a period of time sufficient for delivery ofthe interfering RNA to the cells to occur.

The lipid particles of the invention (e.g., SNALP) can be adsorbed toalmost any cell type with which they are mixed or contacted. Onceadsorbed, the particles can either be endocytosed by a portion of thecells, exchange lipids with cell membranes, or fuse with the cells.Transfer or incorporation of the nucleic acid (e.g., interfering RNA)portion of the particle can take place via any one of these pathways. Inparticular, when fusion takes place, the particle membrane is integratedinto the cell membrane and the contents of the particle combine with theintracellular fluid.

The lipid particles of the invention (e.g., SNALP) can be administeredeither alone or in a mixture with a pharmaceutically acceptable carrier(e.g., physiological saline or phosphate buffer) selected in accordancewith the route of administration and standard pharmaceutical practice.Generally, normal buffered saline (e.g., 135-150 mM NaCl) will beemployed as the pharmaceutically acceptable carrier. Other suitablecarriers include, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like, including glycoproteins for enhanced stability,such as albumin, lipoprotein, globulin, etc. Additional suitablecarriers are described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES,Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As usedherein, “carrier” includes any and all solvents, dispersion media,vehicles, coatings, diluents, antibacterial and antifungal agents,isotonic and absorption delaying agents, buffers, carrier solutions,suspensions, colloids, and the like. The phrase “pharmaceuticallyacceptable” refers to molecular entities and compositions that do notproduce an allergic or similar untoward reaction when administered to ahuman.

The pharmaceutically acceptable carrier is generally added followinglipid particle formation. Thus, after the lipid particle (e.g., SNALP)is formed, the particle can be diluted into pharmaceutically acceptablecarriers such as normal buffered saline.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2 to 5%, to as much as about 10 to 90% by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration.

The pharmaceutical compositions of the present invention may besterilized by conventional, well-known sterilization techniques. Aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, andcalcium chloride. Additionally, the particle suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol, and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

In some embodiments, the lipid particles of the invention (e.g., SNALP)are particularly useful in methods for the therapeutic delivery of oneor more nucleic acids comprising an interfering RNA sequence (e.g.,siRNA). In particular, it is an object of this invention to provide invivo methods for treatment of anemia of inflammation in humans bydownregulating or silencing the transcription and/or translation of oneor more SMAD4 isozymes.

A. In Vivo Administration

Systemic delivery for in vivo therapy, e.g., delivery of a therapeuticnucleic acid to a distal target cell via body systems such as thecirculation, has been achieved using nucleic acid-lipid particles suchas those described in PCT Publication Nos. WO 05/007196, WO 05/121348,WO 05/120152, and WO 04/002453, the disclosures of which are hereinincorporated by reference in their entirety for all purposes. Thepresent invention also provides fully encapsulated lipid particles thatprotect the nucleic acid from nuclease degradation in serum, arenon-immunogenic, are small in size, and are suitable for repeat dosing.

For in vivo administration, administration can be in any manner known inthe art, e.g., by injection, oral administration, inhalation (e.g.,intransal or intratracheal), transdermal application, or rectaladministration. Administration can be accomplished via single or divideddoses. The pharmaceutical compositions can be administered parenterally,i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In some embodiments, thepharmaceutical compositions are administered intravenously orintraperitoneally by a bolus injection (see, e.g., U.S. Pat. No.5,286,634). Intracellular nucleic acid delivery has also been discussedin Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino etal., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. DrugCarrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).Still other methods of administering lipid-based therapeutics aredescribed in, for example, U.S. Pat. Nos. 3,993,754; 4,145,410;4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles canbe administered by direct injection at the site of disease or byinjection at a site distal from the site of disease (see, e.g., Culver,HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71 (1994)). The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

In embodiments where the lipid particles of the present invention (e.g.,SNALP) are administered intravenously, at least about 5%, 10%, 15%, 20%,or 25% of the total injected dose of the particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In other embodiments,more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% ofthe total injected dose of the lipid particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In certain instances,more than about 10% of a plurality of the particles is present in theplasma of a mammal about 1 hour after administration. In certain otherinstances, the presence of the lipid particles is detectable at leastabout 1 hour after administration of the particle. In some embodiments,the presence of a therapeutic nucleic acid such as an interfering RNAmolecule is detectable in cells at about 8, 12, 24, 36, 48, 60, 72 or 96hours after administration. In other embodiments, downregulation ofexpression of a target sequence, such as a viral or host sequence, by aninterfering RNA (e.g., siRNA) is detectable at about 8, 12, 24, 36, 48,60, 72 or 96 hours after administration. In yet other embodiments,downregulation of expression of a target sequence, such as a viral orhost sequence, by an interfering RNA (e.g., siRNA) occurs preferentiallyin infected cells and/or cells capable of being infected. In furtherembodiments, the presence or effect of an interfering RNA (e.g., siRNA)in cells at a site proximal or distal to the site of administration isdetectable at about 12, 24, 48, 72, or 96 hours, or at about 6, 8, 10,12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration. Inadditional embodiments, the lipid particles (e.g., SNALP) of theinvention are administered parenterally or intraperitoneally.

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering nucleic acid compositions directly tothe lungs via nasal aerosol sprays have been described, e.g., in U.S.Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs usingintranasal microparticle resins and lysophosphatidyl-glycerol compounds(U.S. Pat. No. 5,725,871) are also well-known in the pharmaceuticalarts. Similarly, transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045. The disclosures of the above-described patents are hereinincorporated by reference in their entirety for all purposes.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions are preferablyadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically, or intrathecally.

Generally, when administered intravenously, the lipid particleformulations are formulated with a suitable pharmaceutical carrier. Manypharmaceutically acceptable carriers may be employed in the compositionsand methods of the present invention. Suitable formulations for use inthe present invention are found, for example, in REMINGTON′SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). A variety of aqueous carriers may be used, for example,water, buffered water, 0.4% saline, 0.3% glycine, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Generally, normal buffered saline (135-150mM NaCl) will be employed as the pharmaceutically acceptable carrier,but other suitable carriers will suffice. These compositions can besterilized by conventional liposomal sterilization techniques, such asfiltration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. Thesecompositions can be sterilized using the techniques referred to aboveor, alternatively, they can be produced under sterile conditions. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles disclosed herein may bedelivered via oral administration to the individual. The particles maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, pills, lozenges, elixirs,mouthwash, suspensions, oral sprays, syrups, wafers, and the like (see,e.g., U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes). These oral dosage forms may also contain thefollowing: binders, gelatin; excipients, lubricants, and/or flavoringagents. When the unit dosage form is a capsule, it may contain, inaddition to the materials described above, a liquid carrier. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. Of course, any material used inpreparing any unit dosage form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed.

Typically, these oral formulations may contain at least about 0.1% ofthe lipid particles or more, although the percentage of the particlesmay, of course, be varied and may conveniently be between about 1% or 2%and about 60% or 70% or more of the weight or volume of the totalformulation. Naturally, the amount of particles in each therapeuticallyuseful composition may be prepared is such a way that a suitable dosagewill be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

Formulations suitable for oral administration can consist of: (a) liquidsolutions, such as an effective amount of a packaged therapeutic nucleicacid (e.g., interfering RNA) suspended in diluents such as water,saline, or PEG 400; (b) capsules, sachets, or tablets, each containing apredetermined amount of a therapeutic nucleic acid (e.g., interferingRNA), as liquids, solids, granules, or gelatin; (c) suspensions in anappropriate liquid; and (d) suitable emulsions. Tablet forms can includeone or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates,corn starch, potato starch, microcrystalline cellulose, gelatin,colloidal silicon dioxide, talc, magnesium stearate, stearic acid, andother excipients, colorants, fillers, binders, diluents, bufferingagents, moistening agents, preservatives, flavoring agents, dyes,disintegrating agents, and pharmaceutically compatible carriers. Lozengeforms can comprise a therapeutic nucleic acid (e.g., interfering RNA) ina flavor, e.g., sucrose, as well as pastilles comprising the therapeuticnucleic acid in an inert base, such as gelatin and glycerin or sucroseand acacia emulsions, gels, and the like containing, in addition to thetherapeutic nucleic acid, carriers known in the art.

In another example of their use, lipid particles can be incorporatedinto a broad range of topical dosage forms. For instance, a suspensioncontaining nucleic acid-lipid particles such as SNALP can be formulatedand administered as gels, oils, emulsions, topical creams, pastes,ointments, lotions, foams, mousses, and the like.

When preparing pharmaceutical preparations of the lipid particles of theinvention, it is preferable to use quantities of the particles whichhave been purified to reduce or eliminate empty particles or particleswith therapeutic agents such as nucleic acid associated with theexternal surface.

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as primates(e.g., humans and chimpanzees as well as other nonhuman primates),canines, felines, equines, bovines, ovines, caprines, rodents (e.g.,rats and mice), lagomorphs, and swine.

The amount of particles administered will depend upon the ratio oftherapeutic nucleic acid (e.g., interfering RNA) to lipid, theparticular therapeutic nucleic acid used, the disease or disorder beingtreated, the age, weight, and condition of the patient, and the judgmentof the clinician, but will generally be between about 0.01 and about 50mg per kilogram of body weight, preferably between about 0.1 and about 5mg/kg of body weight, or about 108-1010 particles per administration(e.g., injection).

B. In Vitro Administration

For in vitro applications, the delivery of therapeutic nucleic acids(e.g., interfering RNA) can be to any cell grown in culture, whether ofplant or animal origin, vertebrate or invertebrate, and of any tissue ortype. In preferred embodiments, the cells are animal cells, morepreferably mammalian cells, and most preferably human cells.

Contact between the cells and the lipid particles, when carried out invitro, takes place in a biologically compatible medium. Theconcentration of particles varies widely depending on the particularapplication, but is generally between about 1 μmol and about 10 mmolTreatment of the cells with the lipid particles is generally carried outat physiological temperatures (about 37° C.) for periods of time of fromabout 1 to 48 hours, preferably of from about 2 to 4 hours.

In one group of preferred embodiments, a lipid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/ml, more preferably about 2×10⁴ cells/ml.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 0.2 μg/ml, more preferably about 0.1 μg/ml.

To the extent that tissue culture of cells may be required, it iswell-known in the art. For example, Freshney, Culture of Animal Cells, aManual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchleret al., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977), and the references cited thereinprovide a general guide to the culture of cells. Cultured cell systemsoften will be in the form of monolayers of cells, although cellsuspensions are also used.

Using an Endosomal Release Parameter (ERP) assay, the deliveryefficiency of the SNALP or other lipid particle of the invention can beoptimized. An ERP assay is described in detail in U.S. PatentPublication No. 20030077829, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Moreparticularly, the purpose of an ERP assay is to distinguish the effectof various cationic lipids and helper lipid components of SNALP or otherlipid particle based on their relative effect on binding/uptake orfusion with/destabilization of the endosomal membrane. This assay allowsone to determine quantitatively how each component of the SNALP or otherlipid particle affects delivery efficiency, thereby optimizing the SNALPor other lipid particle. Usually, an ERP assay measures expression of areporter protein (e.g., luciferase, β-galactosidase, green fluorescentprotein (GFP), etc.), and in some instances, a SNALP formulationoptimized for an expression plasmid will also be appropriate forencapsulating an interfering RNA. In other instances, an ERP assay canbe adapted to measure downregulation of transcription or translation ofa target sequence in the presence or absence of an interfering RNA(e.g., siRNA). By comparing the ERPs for each of the various SNALP orother lipid particles, one can readily determine the optimized system,e.g., the SNALP or other lipid particle that has the greatest uptake inthe cell.

C. Cells for Delivery of Lipid Particles

The compositions and methods of the present invention are particularlywell suited for treating anemia of inflammation by targeting SMAD4 geneexpression in vivo. The present invention can be practiced on a widevariety of cell types from any vertebrate species, including mammals,such as, e.g, canines, felines, equines, bovines, ovines, caprines,rodents (e.g., mice, rats, and guinea pigs), lagomorphs, swine, andprimates (e.g. monkeys, chimpanzees, and humans).

D. Detection of Lipid Particles

In some embodiments, the lipid particles of the present invention (e.g.,SNALP) are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7, 8 ormore hours. In other embodiments, the lipid particles of the presentinvention (e.g., SNALP) are detectable in the subject at about 8, 12,24, 48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22,24, 25, or 28 days after administration of the particles. The presenceof the particles can be detected in the cells, tissues, or otherbiological samples from the subject. The particles may be detected,e.g., by direct detection of the particles, detection of a therapeuticnucleic acid such as an interfering RNA (e.g., siRNA) sequence,detection of the target sequence of interest (i.e., by detectingexpression or reduced expression of the sequence of interest), detectionof a compound modulated by an EBOV protein (e.g., interferon), detectionof viral load in the subject, or a combination thereof

1. Detection of Particles

Lipid particles of the invention such as SNALP can be detected using anymethod known in the art. For example, a label can be coupled directly orindirectly to a component of the lipid particle using methods well-knownin the art. A wide variety of labels can be used, with the choice oflabel depending on sensitivity required, ease of conjugation with thelipid particle component, stability requirements, and availableinstrumentation and disposal provisions. Suitable labels include, butare not limited to, spectral labels such as fluorescent dyes (e.g.,fluorescein and derivatives, such as fluorescein isothiocyanate (FITC)and Oregon Green™; rhodamine and derivatives such Texas red,tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin,phycoerythrin, AMCA, CyDyes™, and the like; radiolabels such as ³H,¹²⁵I, ³⁵S, 1⁴C, ³²P, ³³P, etc.; enzymes such as horse radish peroxidase,alkaline phosphatase, etc.; spectral colorimetric labels such ascolloidal gold or colored glass or plastic beads such as polystyrene,polypropylene, latex, etc. The label can be detected using any meansknown in the art.

2. Detection of Nucleic Acids

Nucleic acids (e.g., interfering RNA) are detected and quantified hereinby any of a number of means well-known to those of skill in the art. Thedetection of nucleic acids may proceed by well-known methods such asSouthern analysis, Northern analysis, gel electrophoresis, PCR,radiolabeling, scintillation counting, and affinity chromatography.Additional analytic biochemical methods such as spectrophotometry,radiography, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), and hyperdiffusion chromatography may also be employed.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in, e.g., “Nucleic Acid Hybridization, A PracticalApproach,” Eds. Hames and Higgins, IRL Press (1985).

The sensitivity of the hybridization assays may be enhanced through theuse of a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification methods, including the polymerase chain reaction (PCR),the ligase chain reaction (LCR), Qβ-replicase amplification, and otherRNA polymerase mediated techniques (e.g., NASBA™) are found in Sambrooket al., In Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2000); and Ausubel et al., SHORT PROTOCOLS INMOLECULAR BIOLOGY, eds., Current Protocols, Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc. (2002); as well as U.S.Pat. No. 4,683,202; PCR Protocols, A Guide to Methods and Applications(Innis et al. eds.) Academic Press Inc. San Diego, Calif. (1990);Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research,3:81 (1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989);Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell etal., J. Clin. Chem., 35:1826 (1989); Landegren et al., Science, 241:1077(1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene,4:560 (1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan andMalek, Biotechnology, 13:563 (1995). Improved methods of cloning invitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039.Other methods described in the art are the nucleic acid sequence basedamplification (NASBA™, Cangene, Mississauga, Ontario) and Qβ-replicasesystems. These systems can be used to directly identify mutants wherethe PCR or LCR primers are designed to be extended or ligated only whena select sequence is present. Alternatively, the select sequences can begenerally amplified using, for example, nonspecific PCR primers and theamplified target region later probed for a specific sequence indicativeof a mutation. The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

Nucleic acids for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as inhibitor components aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage et al.,Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an automatedsynthesizer, as described in Needham VanD evanter et al., Nucleic AcidsRes., 12:6159 (1984). Purification of polynucleotides, where necessary,is typically performed by either native acrylamide gel electrophoresisor by anion exchange HPLC as described in Pearson et al., J. Chrom.,255:137 149 (1983). The sequence of the synthetic polynucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology, 65:499.

An alternative means for determining the level of transcription is insitu hybridization. In situ hybridization assays are well-known and aregenerally described in Angerer et al., Methods Enzymol., 152:649 (1987).In an in situ hybridization assay, cells are fixed to a solid support,typically a glass slide. If DNA is to be probed, the cells are denaturedwith heat or alkali. The cells are then contacted with a hybridizationsolution at a moderate temperature to permit annealing of specificprobes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters.

IX. EXAMPLES

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1 siRNA-Mediated Reduction of SMAD4 Gene Expression In Vitro

This Example shows siRNA-mediated reduction of SMAD4 gene expression inmouse hepatocyte cells in vitro.

Materials:

All siRNA molecules used in these studies were chemically synthesizedand annealed using standard procedures.

TABLE 1 SMAD family member 4 (SMAD4)-targetingsiRNA sequences used in this study: Identi- Sense StrandAntisense Strand fier (5′ to 3′) (5′ to 3′) 1 rUmGrCrArCrCrArUrArCrArCrrArUrUrAmGrGmUrGmUrGrUr ArCrCrUrArArUmUrU ArUmGrGmUrGrCrArGmU(SEQ ID NO: 3) (SEQ ID NO: 4) 2 mGrCrArCrCrArUrArCrArCrArrArArUrUrArGmGrUmGrUrGm CrCrUrArAmUrUmUrG UrArUmGrGmUrGrCrArG(SEQ ID NO: 5) (SEQ ID NO: 6) 3 rArGmGrUrArGmGrArGrArGrrUrUrArArArCrGmUrCrUrCrUr ArCmGrUmUrUrArArGrG CrCmUrArCrCrUmGrA(SEQ ID NO: 7) (SEQ ID NO: 8) Where ‘r' indicates a ribonucleotide and‘m’ indicates a 2′-O-methylated ribonucleotideMethods:Formulations:

siRNA molecules were encapsulated into serum-stable lipid nanoparticles(LNP) composed of the following lipids: (1) the lipid conjugatePEG2000-C-DMA (3-N—[(-methoxypoly(ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine); (2) a cationiclipid, DLinDMA; (3) the phospholipid DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine) (Avanti Polar Lipids;Alabaster, Ala.); and (4) synthetic cholesterol (Sigma-Aldrich Corp.;St. Louis, Mo.) in the molar ratio 1.4:57.1:7.1:34.3, respectively. Thefinal siRNA/lipid ratio was approximately 0.11 to 0.15 (wt/wt). Uponformation of the siRNA-loaded particles (mean particle sizes were 75-95nm), LNP were filter sterilized through a 0.2 μm filter before use.

Mammalian Cell Treatments and mRNA Analysis:

Primary hepatocytes were isolated from a mouse and maintained asadherent cultures in 96-well plates. SMAD4-targeting or non-targetingcontrol LNP was added to the culture medium at siRNA concentrations of1.25 and 5.0 nM; each treatment condition was performed in triplicatewells. After approximately 24 h LNP incubation, cells were harvested andlysed for mRNA analysis. Mouse SMAD4 mRNA in cell lysate was measuredusing a branched DNA assay (Panomics, Freemont, Calif.; now part ofAffymetrix) with normalization against mouse glyceraldehyde 3-phosphatedehydrogenase (GAPDH).

Results:

Table 2 shows the gene silencing activity of LNP treatments (mean oftriplicate wells, ±standard deviation) described as percentages ofGAPDH-normalized SMAD4 mRNA relative to the amount of GAPDH-normalizedSMAD4 mRNA measured in untreated culture wells arbitrarily set at“100.0%” (mean of 17 replicate wells, ±9.9% standard deviation).

TABLE 2 siRNA 1.25 nM 5 nM 1 (83.1 ± 5.8)% (62.7 ± 4.6)% 2 (47.2 ± 5.4)%(28.6 ± 0.4)% 3 (32.3 ± 0.4)% (10.8 ± 1.9)% Non-targeting control (111.3± 2.4)%  86.7%Conclusion:

All three LNP-formulated siRNA targeting mouse SMAD4 demonstrated genesilencing activity in isolated primary mouse hepatocytes, in adose-responsive manner. Of the three tested, duplex 3 was the mostactive and duplex 1 was the least active. The foregoing LNP-formulatedSMAD4-targeting siRNA induced the desired effect of reducing SMAD4 geneexpression in a type of mammalian cell that is relevant for an intendedtherapeutic use.

Example 2 siRNA-Mediated Reduction of SMAD4 Gene Expression In Vivo

This Example shows siRNA-mediated reduction of SMAD4 gene expression inmouse liver in vivo.

Materials:

Mouse SMAD4 siRNA duplex 2 and 3 are described in Example 1.

Methods:

LNP formulations were prepared as described in Example 1. siRNAs wereencapsulated into serum-stable lipid nanoparticles (LNP) composed of thefollowing lipids: (1) the lipid conjugate PEG2000-C-DMA(3-N—[(-methoxypoly(ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine); (2) a cationiclipid, e.g. dilinoleylmethoxypropyl-(N,N dimethyl)-3-amine (3) thephospholipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) (AvantiPolar Lipids; Alabaster, Ala.); and (4) synthetic cholesterol(Sigma-Aldrich Corp.; St. Louis, Mo.) in the molar ratio 1.5:50:10:38.5,respectively.

Animal Treatments:

Male C57BL/6 mice were obtained from Harlan Labs. Animals wereadministered a single dose of LNP-formulated siRNA or phosphate-bufferedsaline via 10 mL per kg intravenous injection in the lateral tail vein.The administered siRNA dosage was either 0.2, 1.0 or 5.0 mg per kg bodyweight. Approximately 24 h after LNP injection, animals were euthanizedand liver tissue was collected into RNAlater® RNA stabilizing solution.

Tissue Analysis:

Liver tissues were analyzed for mouse SMAD4 mRNA levels normalizedagainst GAPDH mRNA levels using the QuantiGene® branched DNA assay(Panomics, Freemont, Calif.; now part of Affymetrix) essentially asdescribed in Judge et al., 2006, Molecular Therapy 13:494.

Results:

As shown in Table 3, the gene silencing activity of LNP treatments (meanof 4 animals, ±standard deviation) are described as percentage liverGAPDH-normalized SMAD4 mRNA relative to the amount of GAPDH-normalizedSMAD4 liver mRNA measured in control PBS-treated animals arbitrarily setat “100.0%” (mean of 4 animals, ±16.3% standard deviation).

TABLE 3 Liver SMAD4: GAPDH mRNA Ratio Relative to PBS Control TreatmentGroup Mean Formulated siRNA 2, 0.2 mg/kg (49.8 ± 6.3)% Formulated siRNA2, 1.0 mg/kg (37.5 ± 3.9)% Formulated siRNA 2, 5.0 mg/kg (24.6 ± 4.4)%Formulated siRNA 3, 0.2 mg/kg (30.7 ± 2.0)% Formulated siRNA 3, 1.0mg/kg (24.7 ± 5.6)% Formulated siRNA 3, 5.0 mg/kg (20.3 ± 2.2)%Non-targeting control siRNA, 5.0 mg/kg (122.9 ± 18.2)% Conclusion:

A single intravenous administration of LNP-formulated siRNA duplex 2, or3, targeting mouse SMAD4 resulted in SMAD4 gene silencing activity inmice, in a dose-responsive manner. This example shows thatLNP-formulated SMAD4-targeting siRNA induced the desired effect ofreducing SMAD4 gene expression in a whole-animal system via a route ofadministration that is relevant for an intended therapeutic use.

Example 3 Preparation of Serum-Stable Nucleic Acid-Lipid Particles

This Example describes a method for making serum-stable nucleicacid-lipid particles of the present invention.

siRNA molecules are chemically synthesized and annealed using standardprocedures.

In some embodiments, siRNA molecules are encapsulated into serum-stablenucleic acid-lipid particles composed of the following lipids: (1) thelipid conjugate PEG2000-C-DMA (3-N—[(-methoxypoly(ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine); (2) a cationiclipid, e.g. DLinDMA; (3) the phospholipid DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine) (Avanti Polar Lipids;Alabaster, Ala.); and (4) synthetic cholesterol (Sigma-Aldrich Corp.;St. Louis, Mo.) in the molar ratio 1.4:57.1:7.1:34.3, respectively. Inother words, siRNA molecules are encapsulated into nucleic acid-lipidparticles of the following “1:57” formulation: 1.4% PEG2000-C-DMA; 57.1%cationic lipid; 7.1% DPPC; and 34.3% cholesterol. It should beunderstood that the 1:57 formulation is a target formulation, and thatthe amount of lipid (both cationic and non-cationic) present and theamount of lipid conjugate present in the formulation may vary.Typically, in the 1:57 formulation, the amount of cationic lipid is 57.1mol %±5 mol %, and the amount of lipid conjugate is 1.4 mol %±0.5 mol %,with the balance of the 1:57 formulation being made up of non-cationiclipid (e.g., phospholipid, cholesterol, or a mixture of the two).Formulations are made using the process described in United StatesPatent Application US2007/0042031, which is incorporated herein byreference in its entirety. Upon formation the nucleic acid-lipidparticles are dialyzed against PBS and filter sterilized through a 0.2μm filter before use. Mean particle sizes can be in the range of 75-90nm, with final siRNA/lipid ratio of 0.15 (wt/wt).

What is claimed is:
 1. A composition comprising an interfering RNA thatsilences SMAD4 gene expression, wherein the interfering RNA is an siRNAthat comprises a sense strand and a complementary antisense strand,wherein the siRNA is Identifier 3 (SEQ ID NO:7 and SEQ ID NO:8).
 2. Thecomposition of claim 1, further comprising a pharmaceutically acceptablecarrier.
 3. A nucleic acid-lipid particle comprising: (a) an interferingRNA that silences SMAD4 gene expression, wherein the interfering RNA isan siRNA that comprises a sense strand and a complementary antisensestrand, wherein the siRNA is Identifier 3 (SEQ ID NO:7 and SEQ ID NO:8);(b) a cationic lipid; and (c) a non-cationic lipid.
 4. The nucleicacid-lipid particle of claim 3, wherein the cationic lipid comprises1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-di-y-linolenyloxy-N,Ndimethylaminopropane (y-DLenDMA), a saltthereof, or a mixture thereof.
 5. The nucleic acid-lipid particle ofclaim 3, wherein the cationic lipid comprises2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), asalt thereof, or a mixture thereof.
 6. The nucleic acid-lipid particleof claim 3, wherein the cationic lipid comprises(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), dilinoleylmethyl-3-dimethylaminopropionate(DLin-MC2-DMA), a salt thereof, or a mixture thereof.
 7. The nucleicacid-lipid particle of claim 6, wherein the cationic lipid isDLin-M-C3-DMA, a salt thereof.
 8. The nucleic acid-lipid particle ofclaim 3, wherein the non-cationic lipid is a phospholipid.
 9. Thenucleic acid-lipid particle of claim 3, wherein the non-cationic lipidis cholesterol or a derivative thereof.
 10. The nucleic acid-lipidparticle of claim 9, wherein the non-cationic lipid is cholesterol. 11.The nucleic acid-lipid particle of claim 3, wherein the non-cationiclipid is a mixture of a phospholipid and cholesterol or a derivativethereof.
 12. The nucleic acid-lipid particle of claim 3, wherein thephospholipid comprises dipalmitoylphosphatidylcholine (DPPC),distearoylphosphatidylcholine (DSPC), or a mixture thereof.
 13. Thenucleic acid-lipid particle of claim 3, wherein the non-cationic lipidis a mixture of DPPC and cholesterol.
 14. The nucleic acid-lipidparticle of claim 3, further comprising a conjugated lipid that inhibitsaggregation of particles.
 15. The nucleic acid-lipid particle of claim14, wherein the conjugated lipid that inhibits aggregation of particlesis a polyethyleneglycol (PEG)-lipid conjugate.
 16. The nucleicacid-lipid particle of claim 15, wherein the PEG-lipid conjugate isselected from the group consisting of a PEG-diacylglycerol (PEG-DAG)conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, aPEG-phospholipid conjugate, a PEGceramide (PEG-Cer) conjugate, and amixture thereof.
 17. The nucleic acid-lipid particle of claim 15,wherein the PEG-lipid conjugate is a PEG-DAA conjugate.
 18. The nucleicacid-lipid particle of claim 17, wherein the PEG-DAA conjugate isselected from the group consisting of a PEG-didecyloxypropyl (C10)conjugate, a PEG-dilauryloxypropyl (C12) conjugate, aPEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16)conjugate, a PEG-distearyloxypropyl (C18) conjugate, and a mixturethereof.
 19. The nucleic acid-lipid particle of claim 14, wherein theconjugated lipid that inhibits aggregation of particles is apolyoxazoline (POZ)-lipid conjugate.
 20. The nucleic acid-lipid particleof claim 19, wherein the POZ-lipid conjugate is a POZ-DAA conjugate. 21.The nucleic acid-lipid particle of claim 14, wherein the cationic lipidis (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino) butanoate (DLin-M-C3-DMA), the non-cationic lipid ischolesterol, and the conjugated lipid is a a polyethyleneglycol(PEG)-lipid conjugate.
 22. The nucleic acid-lipid particle of claim 3,wherein the interfering RNA is fully encapsulated in the particle. 23.The nucleic acid-lipid particle of claim 3, wherein the particle has alipid:interfering RNA mass ratio of from about 5:1 to about 15:1. 24.The nucleic acid-lipid particle of claim 3, wherein the particle has amedian diameter of from about 30 nm to about 150 nm.
 25. The nucleicacid-lipid particle of claim 3, wherein the cationic lipid comprisesfrom about 50 mol % to about 65 mol % of the total lipid present in theparticle.
 26. The nucleic acid-lipid particle of claim 3, wherein thenon-cationic lipid comprises a mixture of a phospholipid and cholesterolor a derivative thereof, wherein the phospholipid comprises from about 4mol % to about 10 mol % of the total lipid present in the particle andthe cholesterol or derivative thereof comprises from about 30 mol % toabout 40 mol % of the total lipid present in the particle.
 27. Thenucleic acid-lipid particle of claim 26, wherein the phospholipidcomprises from about 5 mol % to about 9 mol % of the total lipid presentin the particle and the cholesterol or derivative thereof comprises fromabout 32 mol % to about 37 mol % of the total lipid present in theparticle.
 28. The nucleic acid-lipid particle of claim 14, wherein theconjugated lipid that inhibits aggregation of particles comprises fromabout 0.5 mol % to about 2 mol % of the total lipid present in theparticle.
 29. A pharmaceutical composition comprising a nucleicacid-lipid particle of claim 3 and a pharmaceutically acceptablecarrier.
 30. A method for introducing an interfering RNA that silencesSMAD4 gene expression into a cell, the method comprising: (a) contactingthe cell with a nucleic acid-lipid particle of claim
 3. 31. The methodof claim 30, wherein the cell is in a mammal.
 32. The method of claim30, wherein the cell is contacted by administering the particle to themammal via a systemic route.
 33. The method of claim 30, wherein themammal is a human.
 34. The method of claim 30, wherein the mammal hasbeen diagnosed with anemia of inflammation.
 35. A method for treatingand/or ameriorating one or more symptoms associated with anemia ofinflammation in a human, the method comprising: (a) administering to thehuman a therapeutically effective amount of a nucleic acid-lipidparticle of claim
 3. 36. The method of claim 35, wherein the particle isadministered via a systemic route.
 37. The method of claim 35, whereinthe human has anemia of inflammation.